JP2005512507A - Use of HMG fragments as anti-inflammatory agents - Google Patents

Abstract

  Disclosed are compositions and methods for inhibiting the release of pro-inflammatory cytokines from vertebrate cells and for inhibiting the inflammatory cytokine cascade in a patient. The composition comprises a vertebrate HMGA box and an antibody preparation that specifically binds to a vertebrate HMGB box. The method includes treating the cell or patient with an amount of the composition sufficient to inhibit pro-inflammatory cytokine release or inhibit the inflammatory cytokine cascade.

Description

Detailed Description of the Invention

This application claims the benefit of US Provisional Application No. 60 / 291,034, filed May 15, 2001. The entirety of these teachings are incorporated herein by reference.

Government Assistance This application, in whole or in part, was assisted by grant number RO1 GM 57226-02 from the National Institutes of Health. The US government has certain rights in this invention.

BACKGROUND OF THE INVENTION In many cases, pro-inflammatory cytokines such as tumor necrosis factor (TNF), interleukin (IL) -1α, IL-1β, IL-6, platelet activating factor (PAF), macrophage migration inhibitory factor ( MIF) and other compounds induce inflammation. Depending on several different cell types, the most important are not only immune cells (eg mononuclear leukocytes, macrophages and neutrophils), but also fibroblasts, osteoblasts, smooth muscle cells, epithelial cells, neurons etc. These non-immune cells produce these proinflammatory cytokines. These proinflammatory cytokines contribute to various disorders during the early stages of the inflammatory cytokine cascade.

  The inflammatory cytokine cascade contributes to the detrimental attributes of many diseases, including inflammation and apoptosis. Included are diseases characterized by both local and systemic reactions, including but not limited to the following. That is, diseases related to the gastrointestinal tract and accompanying tissues (eg, appendicitis, peptic ulcer, gastric ulcer and duodenal ulcer, peritonitis, pancreatitis, ulcerative colitis, pseudomembranous colitis, acute and ischemic colitis, diverticulitis, Laryngitis, achalasia, cholangitis, cholecystitis, celiac disease, hepatitis, Crohn's disease, enteritis and Whipple's disease, etc .; systemic or local inflammatory diseases and conditions (eg, asthma, allergies, anaphylactic shock, immune complexes) Disease, organ ischemia, reperfusion injury, organ necrosis, hay fever, sepsis, sepsis, endotoxin shock, cachexia, abnormal hyperthermia, eosinophilic granuloma, granulomatosis, and sarcoidosis); urogenital system And associated tissue-related diseases (eg, septic abortion, epididymis, vaginitis, prostatitis, and urethritis Diseases related to the respiratory system and accompanying tissues (eg bronchitis, emphysema, rhinitis, cystic fibrosis, pneumonia, adult respiratory distress syndrome, pneumoultramicroscopicsilicovolcanoconiosis, alveolitis, bronchiolitis Diseases such as influenza, RS virus, HIV, hepatitis B virus, hepatitis C virus and herpes virus), bacterial infections, etc. Diseases caused (eg disseminated bacteremia, dengue fever, etc.), diseases caused by fungal infection (eg candidiasis) and diseases caused by protozoa and multicellular parasites (eg malaria, filariasis, amoeba) Dermatitis and cystic cysts); skin diseases and skin conditions (eg burns, dermatitis, skin muscles) , Sunburn, urticaria, warts, and wheal); diseases related to the circulatory system and accompanying tissues (eg, vasulitis, vasculitis, endocarditis, arteritis, atherosclerosis) Disease, thrombophlebitis, epicarditis, congestive heart failure, myocarditis, myocardial ischemia, nodular periarteritis, and rheumatic fever); diseases related to the central or peripheral nervous system and associated tissues (Eg, Alzheimer's disease, meningitis, encephalitis, multiple sclerosis, cerebral infarction, cerebral embolism, Guillame-Barre syndrome, neuritis, neuralgia, spinal cord injury, paralysis, and uveitis); Diseases of joints, muscles and connective tissues (eg, various arthritis and joint pain, osteomyelitis, fasciitis, Paget's disease, gout, periodontal disease, rheumatoid arthritis, and synovitis); other autoimmunity Disease and Inflammatory disorders (eg, myasthenia gravis, thyroiditis, systemic lupus erythematosus, Goodpasture syndrome, Behcet's syndrome, allograft rejection, graft-versus-host disease, type I diabetes, ankylosing spondylitis, Buerger's disease, type I Diabetes, ankylosing spondylitis, Buerger's disease, and Reiter's syndrome); and various cancers, tumors and proliferative disorders (such as Hodgkin's disease); and in any case, an inflammatory response or host immunity to any primary disease It is a response.

  Early pro-inflammatory cytokines (eg TNF, IL-1 etc.) mediate inflammation and induce late release of high mobility group-1 (HMG1) (also known as HMG-1 and HMGB1) To do. This HMG1 is a protein that accumulates in the serum and mediates the induction of late death and further early pro-inflammatory cytokines.

  HMG1 was first identified as a member at the time of the establishment of a family of DNA-binding proteins important for DNA structure and stability, referred to as the high mobility group (HMG). It was identified almost 40 years ago as a ubiquitously expressed nuclear protein that binds to double-stranded DNA regardless of sequence specificity.

  The binding of HMG1 bends the DNA and promotes the formation of a nucleoprotein complex that promotes transcription of the glucocorticoid receptor and RAG recombinase genes, increasing its stability. The HMG1 molecule has three domains: two DNA-binding motifs called the HMG A box and the HMG B box, and the acidic carboxy terminus. In these two HMG boxes, 80 amino acid and L-type domains are highly conserved. The HMG box is also expressed in other transcriptional regulators including RNA polymerase I transcription factor, human upstream binding factor and lymph specific factor.

  There is recent evidence that HMG1 is involved as a cytokine mediator of late death in endotoxemia. In this study, bacterial endotoxin (lipopolysaccharide (LPS)) activated mononuclear leukocytes / macrophages, releasing HMG1 as a late response to activation, resulting in toxic serum HMG1 levels. It has been demonstrated to rise. Even when antibody administration is delayed until after the initial cytokine response, antibodies to HMG1 suppress endotoxin lethality. Like other proinflammatory cytokines, HMG1 is a potent activator of mononuclear leukocytes. Application of HMG1 intratracheally causes acute lung disease and anti-HMG1 antibodies protect against endotoxin-induced pulmonary edema. Serious patients with sepsis or hemorrhagic shock have elevated serum HMG1 levels. And that level is significantly higher in individuals who did not survive compared to those who survived.

  HMG1 is further involved as a ligand for RAGE. RAGE is a receptor for multiple ligands of the immunoglobulin superfamily. RAGE is expressed in endothelial cells, smooth muscle cells, mononuclear leukocytes, and nerves, and ligand interactions transduce signals via MAP kinase, P21 ras, and NF-kB. Although the kinetics of HMG1 appearing during endotoxemia is delayed, HMG1 may be a preferred therapeutic target, but little is known about the molecular mechanisms for HMG1 signaling and toxicity Not.

  Therefore, it is useful to identify the nature of the pro-inflammatory activity of HMG1, and in particular to identify any inhibitory effect of the active domain (s) and other domains that can respond to this activity.

SUMMARY OF THE INVENTION The present invention states that (1) HMG A-box functions as a competitive inhibitor of HMG pro-inflammatory action, and (2) HMG B-box has predominant HMG pro-inflammatory activity. Based on discovery.

  Accordingly, the present invention relates to a polypeptide comprising a vertebrate HMG A box or biologically active fragment thereof, or a polypeptide comprising a non-natural HMG A box or biologically active fragment thereof. The A box of HMG or embodiments thereof can inhibit the release of pro-inflammatory cytokines from vertebrate cells treated with HMG. The HMG A box is preferably a mammalian HMG A box, more preferably a mammalian HMG1 A box, such as a human HMG1 A box, of SEQ ID NO: 4 or SEQ ID NO: 22. Most preferred is the A box of HMG1 comprising or consisting of the sequence. In one preferred embodiment, the vertebrate cell is a mammalian macrophage. The present invention further encompasses vectors encoding these polypeptides.

  In another aspect, the present invention relates to a composition comprising an A box polypeptide of HMG as described above or a biologically active fragment thereof in a pharmaceutically acceptable excipient. In these embodiments, the composition is capable of inhibiting a condition characterized by activation of the inflammatory cytokine cascade. The composition can further comprise an antagonist of early sepsis mediator. As an antagonist of early sepsis mediator, an antagonist of a cytokine selected from the group consisting of TNF, IL-1α, IL-1β, MIF and IL-6 is preferable, an antibody against TNF or MIF, or an antagonist of IL-1 receptor Is more preferable.

  In these embodiments, preferably the condition is appendicitis, peptic ulcer, gastric ulcer and duodenal ulcer, peritonitis, pancreatitis, ulcerative colitis, pseudomembranous colitis, acute colitis and ischemic colitis, diverticulitis, epiglottitis , Achalasia, cholangitis, cholecystitis, hepatitis, Crohn's disease, enteritis, whipple disease, asthma, allergy, anaphylactic shock, immune complex disease, organ ischemia, reperfusion injury, organ necrosis, hay fever, sepsis, sepsis, endotoxin Shock, cachexia, hyperthermia, eosinophilic granuloma, granulomatosis, sarcoidosis, septic abortion, epididymis, vaginitis, prostatitis, urethritis, bronchitis, emphysema, rhinitis, cyst Fibrosis, pneumonia, pneumoconiosis, alveolitis, bronchiolitis, pharyngitis, pleurisy, sinusitis, influenza, RS virus infection, herpes infection, HIV infection, hepatitis B Rus infection, hepatitis C virus infection, disseminated bacteremia, dengue fever, candidiasis, malaria, filariasis, amebiasis, hydatid cyst, burn, dermatitis, dermatomyositis, sunburn, hives, warts, rash, Vasculitis, vasculitis, endocarditis, arteritis, atherosclerosis, thrombophlebitis, epicarditis, myocarditis, myocardial ischemia, nodular periarteritis, rheumatic fever, Alzheimer's disease, Celiac disease, congestive heart failure, adult respiratory distress syndrome, meningitis, encephalitis, multiple sclerosis, cerebral infarction, cerebral embolism, Giant-Barre syndrome, neuritis, neuralgia, spinal cord injury, paralysis, uveitis, arthritis Arthralgia, osteomyelitis, fasciitis, Paget's disease, gout, periodontal disease, rheumatoid arthritis, synovitis, myasthenia gravis, thyroiditis, systemic lupus erythematosus, Goodpasture's syndrome, Behcet's syndrome, allogeneic transfer Rejection, graft versus host disease, I diabetes, ankylosing spondylitis, Berger's disease, I diabetes, ankylosing spondylitis, Berger's disease, is selected from the group consisting of Reiter's syndrome, and Hodgkin's disease. More preferably, the condition is appendicitis, peptic ulcer, gastric ulcer and duodenal ulcer, peritonitis, pancreatitis, ulcerative colitis, pseudomembranous colitis, acute colitis and ischemic colitis, hepatitis, Crohn's disease, asthma, allergy, Anaphylactic shock, organ ischemia, reperfusion injury, organ necrosis, hay fever, sepsis, sepsis, endotoxin shock, cachexia, septic miscarriage, disseminated bacteremia, burns, Alzheimer's disease, celiac disease, congestive heart failure, Selected from the group consisting of adult respiratory enhancement syndrome, cerebral infarction, cerebral embolism, spinal cord injury, paralysis, allograft rejection and graft-versus-host disease; most preferably, the condition is endotoxin shock or allograft rejection . Where the condition is allograft rejection, the composition may further comprise an immunosuppressant used to suppress allograft rejection, preferably cyclosporine.

  In a further aspect, the present invention relates to a purified preparation of an antibody that specifically binds to the B box of a vertebrate high mobility group protein (HMG) but does not specifically bind to a non-B box epitope of HMG. . In these embodiments, the antibody is capable of inhibiting the biological activity of the HMG B box polypeptide, eg, the release of proinflammatory cytokines from vertebrate cells treated with HMG. In a preferred embodiment, the HMG B box is a mammalian HMG B box, eg, a human HMG B box, more preferably a HMG1 B box, most preferably the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 20. B box of HMG1 with In another embodiment, the antibody binds to a specific polypeptide sequence in the B box of HMG1. Wherein this sequence comprises amino acids 1-20 of SEQ ID NO: 20 (SEQ ID NO: 16) or amino acids 1-20 of SEQ ID NO: 5 (SEQ ID NO: 23), or amino acids 1-20 of SEQ ID NO: 20 (SEQ ID NO: 16) or SEQ ID NO: 5 consisting of amino acids 1 to 20 (SEQ ID NO: 23). It is also preferred that the vertebrate cell is a mammalian macrophage. In some embodiments, it is preferred that the antibody is humanized.

  In a further aspect, the invention relates to a composition containing any of the antibody preparations described above in a pharmaceutically acceptable excipient. In these embodiments, the composition is capable of inhibiting conditions characterized by activation of the inflammatory cytokine cascade. These compositions can also conveniently include antagonists of the early sepsis mediators already described. Preferred conditions that are conveniently treated by these compositions are those that are mediated by or characterized by activation of the inflammatory cytokine cascade, eg, with the previously described A box compositions The states are listed.

  Furthermore, the present invention relates to a polypeptide containing a vertebrate HMG B box or biologically active fragment thereof, or a non-natural HMG B box or biologically active fragment thereof, The total length is not included. In these embodiments, the polypeptide is capable of causing release of pro-inflammatory cytokines from vertebrate cells. The polypeptide of these embodiments is preferably an HMG B box, more preferably an HMG1 B box, most preferably the HMG1 B box with the amino acid sequence given as SEQ ID NO: 5 or SEQ ID NO: 20. preferable. In another embodiment, the HMG B box fragment comprises the sequence of SEQ ID NO: 16 or SEQ ID NO: 23, or consists of the sequence of SEQ ID NO: 16 or SEQ ID NO: 23. In a preferred embodiment, the vertebrate cell is a mammalian macrophage. The present invention further encompasses vectors encoding these polypeptides.

  The invention further relates to a method of inhibiting the release of proinflammatory cytokines from mammalian cells. This method comprises a polypeptide composition of the A box or biologically active fragment of the A box, or a B box or Treating the cells with any of the B box biologically active fragment antibody compositions. In these embodiments, the cells are preferably macrophages. Furthermore, the proinflammatory cytokine is preferably selected from the group consisting of TNF, IL-1α, IL-1β, MIF and IL-6. More preferably, the cell is a macrophage and the proinflammatory cytokine is preferably selected from the group consisting of TNF, IL-1α, IL-1β, MIF and IL-6. More preferably, the method treats cells of a patient suffering from a condition characterized by activation of the inflammatory cytokine cascade or cells of a patient at risk for that condition. Preferred conditions have already been listed.

  In a related aspect, the invention relates to a method of treating a patient condition characterized by activation of an inflammatory cytokine cascade. This method comprises a polypeptide composition of an A box or biologically active fragment of an A box, or a biologically active B box or B box as described above, in an amount sufficient to inhibit the inflammatory cytokine cascade. Administering to the patient any active fragment antibody composition. Preferred conditions have already been listed.

  A further aspect relates to a method of stimulating the release of proinflammatory cytokines from cells. This method comprises a B box polypeptide or biologically active fragment thereof in an amount sufficient to stimulate the release of proinflammatory cytokines, or a biological activity of the B box polypeptide or B box described above. Treating the cells with a vector of the appropriate fragment. In a related aspect, the present invention relates to a method of effectively reducing a patient's weight or treating obesity. The method comprises administering to the patient an effective amount of a B-box polypeptide of HMG or a biologically active fragment thereof. In one embodiment, the HMG B box polypeptide or biologically active fragment thereof is in a pharmaceutically acceptable excipient.

  The invention further relates to a method for determining whether a compound inhibits inflammation. The method comprises (a) a cell that releases a pro-inflammatory cytokine when exposed to a B box of vertebrate HMG or a biologically active fragment thereof; and (b) a B box or biology of HMG. Combining the compound with a chemically active fragment thereof, and then determining whether the compound inhibits the release of pro-inflammatory cytokines from the cell. Preferably, the HMG B box is a mammalian HMG B box, eg, a HMG1 B box. Preferred pro-inflammatory cytokines have already been described.

DETAILED DESCRIPTION OF THE INVENTION In carrying out the present invention, standard techniques of cell culture, molecular biology, microbiology, cell biology, and immunology are employed unless otherwise indicated. These techniques are well known to those skilled in the art. Such techniques are explained fully in the literature. For example, Sambrook et al., 1989, “Molecular Cloning: A Laboratory Manual”, Cold Spring Harbor Laboratory Press; Ausubel et al. (1995), “Short Protocols in Molecular Biology”, John WILey and Sons; Methods in Enzymology (several volumes); See in Cell Biology (several volumes) and Methods in Molecular Biology (several volumes).

  The present invention is based on a series of discoveries that further elucidate the various properties of HMG1's ability to induce the production of pro-inflammatory cytokines and inflammatory cytokine cascades. Specifically, the pro-inflammatory active domain of HMG1 is the B box (and in particular, the first 20 amino acids of the B box), and the B box specific antibody is responsible for the release of pro-inflammatory cytokines and It has been discovered that inhibiting the inflammatory cytokine cascade can result in alleviating the harmful symptoms caused by the inflammatory cytokine cascade. Furthermore, it has also been discovered that the A box is a weak agonist of inflammatory cytokine release and competitively inhibits the pro-inflammatory activity of B box and HMG1.

  As used herein, an “HMG polypeptide” or “HMG protein” is one that is substantially pure or a polypeptide that is substantially pure and isolated. Here, the isolated polypeptide is a polypeptide that has been separated from components that naturally accompany the polypeptide, or a recombinantly produced polypeptide having the same amino acid sequence. Intensify inflammation and / or increase the release of pro-inflammatory cytokines from cells and / or enhance the activity of the inflammatory cytokine cascade. In one embodiment, the HMG polypeptide has one of the above biological activities. In another embodiment, the HMG polypeptide has two of the above biological activities. In a third embodiment, the HMG polypeptide has all three of the above biological activities.

  The HMG polypeptide is preferably a mammalian HMG polypeptide, such as a human HMG1 polypeptide. Preferably, the HMG polypeptide has at least 60% sequence identity to a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 18, at least 70%, More preferably, it has 75%, 80%, 85%, or 90% sequence identity and most preferably has at least 95% sequence identity. Here, sequence identity is determined using the BLAST program and the parameters described herein. Examples of HMG polypeptides include polypeptides that contain or consist of the sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 18. Preferably, the HMG polypeptide contains a B-box DNA binding domain and / or an A-box DNA binding domain as described herein, and / or an acidic carboxy terminus. Other examples of HMG polypeptides are described in GenBank accession numbers AAA64970, AAB08987, P07155, AAA20508, S29857, P09429, NP_002119, CAA31110, the entire teachings of which are incorporated herein by reference. Incorporated. Further examples of HMG polypeptides include mammalian HMG1, HMG2, HMG-2A, HMG14, HMG17, HMG I and HMGY; non-mammalian HMG T1 and HMG T2 (rainbow trout), HMG-X (Xenopus laevis), HMG D / Z (Drosophila), yeast polypeptide NHP10 protein (HMG protein homolog NHP 1) and non-histone chromosomal proteins; HMG 1 / 2-like proteins (wheat, corn, soybean); upstream binding factor (UBF-1), Single-strand recognition protein (SSRP) or structure-specific recognition protein; HMG homolog TDP-1; mammalian sex-determining region Y protein (SRY, testis determinant); fungal proteins: mat-1, ste 11 and Mc 1; SOX 14 (and SOX 1-3, 6, 8, 10, 12, and 21); Lymph specific factor (LEF-1); T cell specific transcription factor (TCF-1); and SP100-HMG nuclear self Include but are not limited to antigens. Is not something

  As used herein, “A box of HMG”, also referred to herein as “A box”, is either substantially pure or substantially pure and isolated poly. It is a peptide. As used herein, an isolated polypeptide is one that is separated from components that naturally accompany the polypeptide, consists of an amino acid sequence that is shorter than the full length of the HMG polypeptide, and Biological activity of: having one or more of inhibiting inflammation and / or inhibiting the release of pro-inflammatory cytokines from cells and / or reducing the activity of the inflammatory cytokine cascade is there. In one embodiment, the HMG A box polypeptide has one of the biological activities described above. In another embodiment, the HMG A-box polypeptide has two of the above biological activities. In a third embodiment, the HMG A box polypeptide has all three of the above biological activities. Preferably, the HMG A box has only 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the biological activity of full length HMG. In one embodiment, the amino acid in the A box of HMG consists of the sequence of SEQ ID NO: 4 or SEQ ID NO: 22, or the amino acid sequence in the corresponding region of the HMG protein in a mammal. The HMG A box is also a recombinantly produced polypeptide having the same amino acid sequence as the A box sequence described above. Preferably, the HMG A box is a mammalian HMG A box, eg, a human HMG1 A box. The HMG A box polypeptide of the present invention preferably contains or consists of the sequence of SEQ ID NO: 4 or SEQ ID NO: 22, or the amino acid sequence in the corresponding region of the HMG protein in mammals. Often, the HMG A box has less than about 85 amino acids and more than about 4 amino acids. Examples of polypeptides having an A box sequence therein include HMG1, HMG2, HMG4; structure-specific recognition protein (SSRP); PMS1 protein homolog 1; SOX-1 protein, SOX-2 protein, and SOX-14 A protein; as well as MTT1, but is not limited thereto. It is possible to determine and isolate the sequence of the A box in such a polypeptide by methods described herein, such as sequence comparison with the A box described herein and testing for biological activity. is there.

  The invention further features a non-natural HMG A box. It is preferred that the non-natural HMG A box has at least 60% sequence identity to the sequence of SEQ ID NO: 4 or SEQ ID NO: 22, at least 70%, 75%, 80%, 85%, or 90 It is more preferred to have% sequence identity and most preferred to have at least 95% sequence identity. Here, sequence identity is determined using the BLAST program and one or more of the parameters described herein and the biological activity of the HMG A box.

  The invention further features a biologically active fragment of the A box. “A-box fragment with A-box biological activity” or “A-box biologically-active fragment” refers to an A of HMG having the activity of an A-box of HMG as described herein. Means a fragment of a box. For example, A-box fragments can reduce the release of pro-inflammatory cytokines from vertebrate cells, reduce inflammation, and / or reduce the activity of the inflammatory cytokine cascade. Standard molecular biology techniques can be used to generate the A-box fragment. The fragment can then be determined, for example, by utilizing the methods described herein, to determine whether the fragment inhibits pro-inflammatory cytokine release from the cell when administered to the cell. Evaluate the function. Biologically active fragments of the A box are characterized by the full length of the A box polypeptide, for example, inhibiting the release of pro-inflammatory cytokines from cells or activating the inflammatory cytokine cascade It can be used in the methods described herein for the treatment of patients presenting with symptoms.

  As used herein, an “HMG B box”, also referred to herein as a “B box”, is a substantially pure or substantially pure and isolated poly. It is a peptide. Here, this isolated polypeptide is one that has been separated from components that naturally accompany the polypeptide, consists of an amino acid sequence that is shorter than the full length of the HMG polypeptide, and Biological activity: one or more of enhancing inflammation, increasing the release of pro-inflammatory cytokines from cells, and / or increasing the activity of the inflammatory cytokine cascade. In one embodiment, the HMG B box polypeptide has one of the above biological activities. In another embodiment, the HMG B box polypeptide has two of the above biological activities. In a third embodiment, the HMG B box polypeptide has all three of the above biological activities. It is preferred that the B box of HMG has at least 25%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the biological activity of full length HMG. In another embodiment, the HMG B box does not include the HMG A box. In another embodiment, the B box of HMG is about 90%, 80%, 70%, 60%, 50%, 40%, 35%, 30%, 25%, or 20 of the full length of the HMG1 polypeptide. % Polypeptide. In another embodiment, the box of HMG contains or consists of the sequence of SEQ ID NO: 5 or SEQ ID NO: 20, or the amino acid sequence in the corresponding region of the HMG protein in a mammal, but the full length of the HMG polypeptide. Even shorter than. The HMG B box polypeptide is also a recombinantly produced polypeptide having the same amino acid sequence as the B box sequence described above. Preferably, the HMG B box is a mammalian HMG B box, eg, a human HMG1 B box. Often, the HMG B box has less than about 85 amino acids and more than about 4 amino acids. Examples of polypeptides having a B box sequence therein include HMG polypeptides described herein; single chain recognition protein (SSRP) or structure-specific recognition protein; yeast NHP10 protein (HMG protein homolog NHP1 ); HMG homolog TDP-1; sex determining region Y protein (testis determinant); SOX 14 (and SOX 1-3, 6, 8, 10, 12 and 21); lymph specific factor (LEF-1); Examples include, but are not limited to, T cell specific transcription factor (TCF-1). Using the methods described herein to determine and isolate the sequence of the B box in such a polypeptide, for example by sequence comparison with the B box described herein and testing for biological activity. Is possible.

  The invention further encompasses non-natural HMG B box polypeptides. The non-natural HMG B box polypeptide preferably has at least 60% sequence identity to the sequence of SEQ ID NO: 5 or SEQ ID NO: 20 and is at least 70%, 75%, 80%, 85% Or more preferably 90% sequence identity and most preferably at least 95% sequence identity. Here, sequence identity is determined using the BLAST program as well as the parameters described herein. The B box of HMG preferably consists of the sequence of SEQ ID NO: 5 or SEQ ID NO: 20, or the amino acid sequence in the corresponding region of the HMG protein in mammals.

  In other embodiments, the present invention relates to a vertebrate HMG B box or a fragment thereof having biological activity of a B box, or a polypeptide that comprises a non-natural HMG B box, but not a full-length HMG. “B-box fragment with B-box biological activity” or “B-box biologically-active fragment” means an HMG B-box fragment with an HMG B-box activity. For example, a fragment of the B box can induce the release of pro-inflammatory cytokines from vertebrate cells, intensify inflammation, or induce an inflammatory cytokine cascade. An example of such a B box fragment is a fragment (SEQ ID NO: 16 or SEQ ID NO: 23) comprising the first 20 amino acids of the B box of HMG1 as described herein. Using standard molecular biology techniques, it is possible to generate fragments of the B box. And, when administered to a cell, whether this fragment increases the release of pro-inflammatory cytokines from that cell, for example, compared to an appropriate control using the methods described herein. By determining, the function of this fragment is evaluated.

  As used herein, a “cytokine” is a soluble protein or peptide that is naturally produced by mammalian cells and acts in vivo as a humoral regulator at micromolar to picomolar concentrations. Cytokines can modulate the functional activity of individual cells and tissues either under normal or pathological conditions. Pro-inflammatory cytokines are the ability to cause any of the following physiological responses associated with inflammation: vasodilation, hyperemia, increased vascular permeability associated with edema, granulocyte and mononuclear phagocyte accumulation, or fibrin deposition It has a cytokine. In some cases, pro-inflammatory cytokines can also cause apoptosis, for example in chronic heart failure. Here, TNF is shown to stimulate cardiomyocyte apoptosis (Pulkki, Ann. Med. 29: 339-343, 1997; and Tsutsui et al., Immunol. Rev. 174: 192-209, 2000).

  Non-limiting examples of proinflammatory cytokines include tumor necrosis factor (TNF), interleukin (IL) -1α, IL-1β, IL-6, IL-8, IL-18, interferon γ, HMG-1, Platelet activating factor (PAF) and macrophage migration inhibitory factor (MIF).

  Pro-inflammatory cytokines can be distinguished from anti-inflammatory cytokines that are not mediators of inflammation, such as IL-4, IL-10, and IL-13.

  In many cases, pro-inflammatory cytokines are produced in the inflammatory cytokine cascade. This is defined herein as the in vivo release of at least one pro-inflammatory cytokine in a mammal, where the physiological state of the mammal is affected by the release of the cytokine. Thus, in aspects of the invention where the release of pro-inflammatory cytokines causes a physiological condition that is detrimental to the mind and body, the inflammatory cytokine cascade is inhibited.

  The HMG A box and the HMG B box, which are either natural or non-natural, include polypeptides having sequence identity with the HMG A box and the HMG B box described above. As used herein, two polypeptides (or polypeptides) when the amino acid sequences are at least about 60%, 70%, 75%, 80%, 85%, 90% or 95% homologous or identical. Are substantially homologous or identical. The percent identity of two amino acid sequences (or two nucleic acid sequences) by aligning the sequences so that the intent of the comparison is optimal (eg, gaps may be introduced in the sequence of the first sequence) Can be determined. The amino acids or nucleotides at the corresponding positions are then compared, and the percent identity between the two sequences is a function of the number of identical positions shared by those sequences (ie,% identity = identical positions Number / total number of positions × 100). In certain embodiments, the length of the HMG polypeptide, the length of the HMG A-box polypeptide, or the length of the HMG B-box polypeptide, aligned for purposes of comparison, is a reference sequence (eg, 12A-12E, and the sequences of SEQ ID NOs: 18, 20, and 22), at least 40% is preferred, at least 60% is more preferred, and at least 70% 80%, 90%, or 100% is more preferable. The two sequences can actually be compared by well-known methods, such as those utilizing mathematical algorithms. A preferred, non-limiting example of such a mathematical algorithm is described by Karlin et al., Proc. Natl. Acad. Sci. USA, 90: 5873-5877 (1993). Such an algorithm is incorporated into the BLASTN and BLASTX programs (version 2.2) described in Schaffer et al., Nucleic Acids Res., 29: 2994-3005 (2001). When using the BLAST program and the Gapped BLAST program, the default parameters of the respective programs (for example, BLASTN) can be used. See http://www.ncbi.nlm.nih.gov available on April 10, 2002. In one embodiment, the database to be searched is a non-redundancy (NR) database and no filter parameters for sequence comparison; expectation value 10; word length 3; matrix is BLOSUM62; and gap cost is gap start cost 11 and the gap extension cost may be set to 1.

  Another preferred, non-limiting example of a mathematical algorithm utilized for sequence comparison is the algorithm of Myers and MILler, CABIOS (1989). Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG (Accelrys) sequence alignment software package. When using the ALIGN program to compare amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 may be used. Additional algorithms for sequence analysis are known in the art and are described in Torellis and Robotti, Comput. Appl. Biosci., 10: 3-5 (1994); ADVANCE and ADAM; and Pearson and Lipman, Proc. Natl. Acad. Sci USA, 85: 2444-8 (1988).

  In another embodiment, using the GAP program in the GCG software package (available at http://www.accelrys.com available from August 31, 2001), the Blossom 63 matrix or PAM 250 matrix Alternatively, gap weights 12, 10, 8, 6, or 4 and length weights 2, 3, or 4 can be used to determine the percent identity between two amino acid sequences. In yet another embodiment, using the GAP program in the GCG software package (available at http://www.cgc.com), two nucleic acid sequences with gap weight 50 and length weight 3 The percent identity between them can be determined.

A-box polypeptides and biologically active fragments thereof As noted above, the present invention is capable of inhibiting the release of pro-inflammatory cytokines from vertebrate cells treated with HMG, or of an inflammatory cytokine cascade It relates to a polypeptide composition containing a vertebrate HMG A box, or a biologically active fragment thereof, which can be used to treat a condition characterized by activation.

  When referring to the effect of any composition or method of the present invention on the release of pro-inflammatory cytokines, the use of the terms “inhibit” or “reduce” is at least insignificant but not negligible. Reduction of sex cytokine release is included. In preferred embodiments, pro-inflammatory cytokine release is inhibited by at least 20% over untreated controls; in more preferred embodiments, inhibition is at least 50%; in more preferred embodiments, inhibition is at least 70%; And in the most preferred embodiment, the inhibition is at least 80%. Such a decrease in pro-inflammatory cytokine release can reduce the psychosomatic adverse effects of the inflammatory cytokine cascade in an in vivo manner.

  Because all vertebrate HMG A-boxes exhibit a high degree of sequence conservation (see, eg, Figure 13 for amino acid sequence comparisons of rat, mouse, and human HMG polypeptides), It is believed that the A box can inhibit the release of pro-inflammatory cytokines from vertebrate cells treated with HMG. Therefore, any vertebrate HMG A box is within the scope of the present invention. The HMG A box is preferably a mammalian HMG A box, such as a human HMG1 A box as defined herein as SEQ ID NO: 4 or SEQ ID NO: 22. There is. Furthermore, HMG1 A-box fragments having the biological activity of HMG A-box as described herein are also encompassed by the present invention.

  A non-natural HMG A box (or biologically active fragment thereof) that inhibits the release of pro-inflammatory cytokines from vertebrate cells treated with vertebrate HMG without undue experimentation Those skilled in the art will also recognize that they can be made. Create these non-naturally functional A boxes by aligning the amino acid sequences of the A box of HMG from different sources and substituting one or more of the sequences at the amino acid positions where the A box is different. it can. This substitution is preferably performed using the same amino acid residue present in the A box being compared. Alternatively, conservative substitutions are made from either residue.

  Conservative amino acid substitution refers to the interchangeability of residues with similar side chains. Amino acids with conservative substitutions can be grouped according to their side chain chemistry. For example, one group of amino acids includes amino acids having neutral and hydrophobic side chains (a, v, l, i, p, w, f, and m); Are amino acids (g, s, t, y, c, n, and q) that have sex and polar side chains; another group is amino acids (k, r, and h) that have basic side chains. Another group is an amino acid having an acidic side chain (d and e); another group is an amino acid having an aliphatic side chain (g, a, v, l, and i); A group is amino acids (s and t) having aliphatic-hydroxyl side chains; another group is amino acids having amine-containing side chains (n, q, k, r, and h); Are groups of amino acids (f, y, and w) having aromatic side chains ; And another group is an amino acid having a sulfur-containing side chains (c and m). Preferred groups of conservative amino acid substitutions are: rk; ed, yf, lm; vi, and qh.

  Conservative amino acid substitutions can be expected to maintain the biological activity of the HMG A box polypeptide, whereas the following are the human HMG1 A box (SEQ ID NO: 4) and human HMG2 A An example of how a non-natural A-box polypeptide can be made by comparing the 32-85 residues of SEQ ID NO: 3 of the box (SEQ ID NO: 17).

HMG1 pdasvnfsef skkcserwkt msakekgkfe dmakadkary eremktyipp kget
HMG2 pdssvnfaef skkcserwkt msakekskfe dmaksdkary dremknyvpp kgdk

  For example, by replacing the alanine (a) residue at the third position in the A box of HMG1 with the serine (s) residue present at the third position of the A box of HMG2 A box can be made. One skilled in the art knows that this substitution provides a non-natural functional A box. This is because the s residue functions at a position in the A box of HMG2. Alternatively, the third position of the A box of HMG1 is replaced by any amino acid that is conserved with respect to alanine or serine, such as glycine (g), threonine (t), valine (v) or leucine (l). be able to. Those skilled in the art will recognize that these conservative substitutions can be expected to result in a functional A box. This is because the third position of the A box is not unchanged, so a conservative substitution provides an appropriate structural substitution for the natural amino acid at that position.

  By following the above method, a very large number of non-naturally occurring HMG A-boxes that could be expected to function can be made without undue experimentation, and any particular non-naturally occurring HMG The functionality of the A box can be predicted with sufficient accuracy. In any case, the functionality of any non-naturally occurring HMG A box can be determined by simply adding it to the cells together with HMG without undue experimentation, For example, the methods described herein can be used to determine whether to inhibit the release of pro-inflammatory cytokines by cells.

  The cell in which the release of HMG-induced proinflammatory cytokines inhibited by A box or a biologically active fragment of A box is any cell that can be induced to produce proinflammatory cytokines. Good. In preferred embodiments, the cells are immune cells such as macrophages, monocytes, or neutrophils. In the most preferred embodiment, the cell is a macrophage.

  Polypeptides containing A box or biologically active fragments of A box that can inhibit the production of any one proinflammatory cytokine now known or later discovered are within the scope of the present invention. Preferably, the antibody can inhibit the production of TNF, IL-1β, or IL-6. Most preferably, the antibody can inhibit the production of any pro-inflammatory cytokine produced by vertebrate cells.

  The present invention also relates to a composition containing any of the above polypeptides in a pharmaceutically acceptable excipient. In these embodiments, the composition may inhibit a condition characterized by activation of the inflammatory cytokine cascade. This condition can be a condition in which the inflammatory cytokine cascade causes a systemic reaction involving, for example, endotoxin shock. Alternatively, this condition can be mediated by a local inflammatory cytokine cascade as in rheumatoid arthritis. Non-limiting examples of conditions that can be usefully treated using the present invention include the conditions listed in the background section of this specification. Preferably, the condition is appendicitis, peptic ulcer, gastric or duodenal ulcer, peritonitis, pancreatitis, ulcerative colitis, pseudomembranous colitis, acute colitis or ischemic colitis, diverticulitis, epiglottitis, achalasia, cholangitis , Cholecystitis, hepatitis, Crohn's disease, enteritis, whipple disease, asthma, allergy, anaphylactic shock, immune complex disease, organ ischemia, reperfusion injury, organ necrosis, hay fever, sepsis, sepsis, endotoxin shock, cachexia , Abnormal hyperthermia, eosinophilic granulomas, granulomatosis, sarcoidosis, septic abortion, epididymis, vaginitis, prostatitis, urethritis, bronchitis, emphysema, rhinitis, cystic fibrosis, pneumonia , Pneumoconiosis, alvealitis, bronchiolitis, pharyngitis, pleurisy, sinusitis, influenza, RS virus infection, herpes infection, HIV infection, hepatitis B virus Infection, hepatitis C virus infection, disseminated bacteremia, dengue fever, candidiasis, malaria, filariasis, amebiasis, hydatid cyst, burn, dermatitis, dermatomyositis, sunburn, hives, warts, wheal, pulse Vasculitis, vasculitis, endocarditis, arteritis, atherosclerosis, thrombophlebitis, epicarditis, myocarditis, myocardial ischemia, nodular periarteritis, rheumatic fever, Alzheimer's disease, celiac Disease, congestive heart failure, adult respiratory distress syndrome, meningitis, encephalitis, multiple sclerosis, cerebral infarction, cerebral embolism, Giant-Barre syndrome, neuritis, neuralgia, spinal cord injury, paralysis, uveitis, arthritis, Arthralgia, osteomyelitis, fasciitis, Paget's disease, gout, periodontal disease, rheumatoid arthritis, synovitis, myasthenia gravis, thryoiditis, systemic lupus erythematosus, Goodpascher's syndrome, Behcet's syndrome, Seed transplant rejection, graft versus host disease, I diabetes, ankylosing spondylitis, Berger's disease, I diabetes, ankylosing spondylitis, Reiter (Retier) syndrome or Hodgkin's disease. In a more preferred embodiment, the condition is appendicitis, peptic ulcer, gastric or duodenal ulcer, peritonitis, pancreatitis, ulcerative colitis, pseudomembranous colitis, acute or ischemic colitis, hepatitis, Crohn's disease, asthma, allergy Anaphylactic shock, organ ischemia, reperfusion injury, organ necrosis, hay fever, sepsis, sepsis, endotoxin shock, cachexia, septic abortion, disseminated bacteremia, burn, Alzheimer's disease, celiac disease, congestive heart failure , Adult respiratory syndrome, cerebral infarction, cerebral embolism, spinal cord injury, paralysis, allograft rejection or graft-versus-host disease. In the most preferred embodiment, the condition is endotoxin shock or allograft rejection. Where the condition is allograft rejection, advantageous compositions also include immunosuppressants such as cyclosporine used to suppress allograft rejection.

  The excipient included with the polypeptide in these compositions is selected based on the expected route of administration of the composition in therapeutic applications. The route of administration of the composition depends on the condition to be treated. For example, intravenous injection is preferred for treating systemic disorders such as endotoxin shock, and oral administration is preferred for treating gastrointestinal disorders such as gastric ulcers. The route of administration and dosage of the composition to be administered can be determined by those skilled in the art in the context of standard dose-response studies without undue experimentation. Relevant circumstances to be considered when making these decisions include the condition being treated, the choice of composition to be administered, the age, weight, and individual patient response, and the severity of the patient's symptoms. Thus, depending on the condition, the antibody composition can be administered to the patient orally, parenterally, intranasally, vaginally, rectally, lingually, sublingually, buccally, buccally and transdermally. It can be administered dermally.

  Thus, compositions designed for oral, lingual, sublingual, buccal and buccal administration can be prepared by means well known in the art, for example, inert, without undue experimentation. It can be made using a diluent or edible carrier. The composition can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapy, the pharmaceutical composition of the present invention can be mixed with excipients and used in the form of tablets, troches, capsules, elixirs, suspensions, syrups, cachets, chewing gums and the like. .

  Tablets, pills, capsules, lozenges and the like can also contain binders, recipients, disintegrating agents, lubricants, sweetening agents, and flavoring agents. Some examples of binders include microcrystalline cellulose, gum tragacanth or gelatin. Examples of excipients include starch or lactose. Examples of the disintegrant include alginic acid and corn starch. Examples of lubricants include magnesium stearate or potassium stearate. An example of a glidant is colloidal silicon dioxide. Some examples of sweeteners include sucrose, saccharin and the like. Examples of the corrigent include peppermint, methyl salicylate, orange flavor and the like. The materials used in preparing these various compositions should be pharmaceutically pure and non-toxic in the amounts used.

  The composition of the present invention can be easily administered parenterally, for example, by intravenous injection, intramuscular injection, intrathecal injection or subcutaneous injection. Parenteral administration can be accomplished by mixing the antibody composition of the invention in a solution or suspension. Such solutions or suspensions may also include sterile diluents such as water for injection, physiological saline solution, fixed oil, polyethylene glycol, glycerin, propylene glycol or other synthetic solvents. Parenteral formulations may also contain antibacterial agents such as benzyl alcohol or methyl paraben, antioxidants such as ascorbic acid or sodium bisulfite, and chelating agents such as EDTA. Buffers such as acetate, citrate or phosphate and tonicity adjusting agents such as sodium chloride or glucose can also be added. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Rectal administration includes administering the pharmaceutical composition into the rectum or large intestine. This can be done using suppositories or enemas. Suppository formulations can easily be made by methods known in the art. For example, a suppository formulation may be prepared by heating glycerin to about 120 ° C., dissolving the antibody composition in glycerin, mixing the heated glycerin, and then adding purified water, and adding the hot mixture to the suppository. Can be dispensed by pouring into a mold.

  Transdermal administration includes percutaneous absorption of the composition via the skin. Transdermal formulations include patches, ointments, creams, gels, ointments and the like.

  The present invention involves nasally administering to a mammal a therapeutically effective amount of the composition. As used herein, nasal or nasal administration includes administering the composition to the nasal passages or nasal mucosa of the patient. As used herein, pharmaceutical compositions for nasal administration of a composition can be prepared by well-known methods for administration, for example, nasal sprays, nasal drops, suspensions, gels, ointments, creams or As a powder, a prepared therapeutically effective amount of an agonist is included. Administration of the composition can also be performed using a nasal tampon or nasal sponge.

  The polypeptide compositions described herein can also include antagonists of early sepsis mediators. As used herein, early sepsis mediators are pro-inflammatory cytokines that are released from cells immediately (ie within 30-60 minutes) after induction of the inflammatory cytokine cascade (eg, exposure to LPS). is there. Non-limiting examples of these cytokines are TNF, IL-1α, IL-1β, IL-6, PAF, and MIF. Also included as early sepsis mediators are receptors for these cytokines (eg, tumor necrosis factor receptor type 1) and enzymes required for production of these cytokines (eg, interleukin-1β converting enzyme). Any early sepsis mediator antagonist now known or later discovered may be useful for these embodiments by further inhibiting the inflammatory cytokine cascade.

  Non-limiting examples of early sepsis mediator antagonists include antisense compounds that bind to and interfere with the expression of early sepsis mediator mRNA (eg, Ojwang et al., Biochemistry 36: 6033-6045, 1997; Parnpfer et al. Biol. Reprod. 52: 1316-1326, 1995; US Pat. No. 6,228,642; Yahata et al., Antisense Nucleic Acid Drug Dev. 6: 55-61, 1996; and Taylor et al., Antisense Nucleic Acid Drug Dev. 8: 199- 205, 1998), ribozymes that specifically cleave early sepsis mediator mRNA (eg Leavitt et al., Antisense Nucleic Acid Drug Dev. 10: 409-414, 2000; Kisich et al., 1999; and Hendrix et al., Biochem. J. 314 (Part 2): 655-661, 1996) and antibodies that bind to early sepsis mediators and inhibit their action (eg Kam and Targan, Expert Opin. Pharmacother. 1: 615-622, 2000; Nagahira et al., J. Immunol. Methods 222, 83-92, 1999; Lavine , J Cereb Blood Flow Metab 18:... 52-58, 1998; and Holmes et al., Hybridoma 19: 363-367, there is a reference 2000). Any antagonist of early sepsis mediator now known or later discovered is contemplated as being within the scope of the present invention. One of ordinary skill in the art can determine the amount of early sepsis mediator to use in these compositions to inhibit any particular inflammatory cytokine cascade by routine dose-response studies without undue experimentation. .

B-box polypeptides, biologically active fragments thereof, and antibodies thereof As noted above, the present invention relates to vertebrates that can increase the release of pro-inflammatory cytokines from vertebrate cells treated with HMG. It relates to a polypeptide composition containing the B box of HMG or a biologically active fragment thereof.

  When referring to the effect of any of the compositions or methods of the invention on the release of proinflammatory cytokines, the use of the term “increase” encompasses at least a slight but non-negligible increase in proinflammatory cytokine release. . In preferred embodiments, pro-inflammatory cytokine release is increased by at least 1.5 fold, at least 2 fold, at least 5 fold, or at least 10 fold over untreated controls. Such an increase in pro-inflammatory cytokine release can increase the effect of the inflammatory cytokine cascade in in vivo embodiments. Such polypeptides can also be used to induce weight loss and / or to treat obesity.

  Because all HMG B-boxes exhibit a high degree of sequence conservation (see, eg, Figure 13 for amino acid sequence comparison of rat, mouse, and human HMG polypeptides), a functional, non-naturally occurring A As discussed above with respect to box production, a functional, non-naturally occurring HMG B box is a naturally occurring spine by making substitutions of one or more conserved amino acids or from different sources. It is believed that by comparing animal B-boxes and substituting similar amino acids, it can be made without undue experimentation. In a particularly preferred embodiment, the B box is the sequence of the B box of human HMG1 (two different lengths) or is a fragment of the B box of HMG having the biological activity of the B box SEQ ID NO: 5 Or SEQ ID NO: 20. For example, the 20 amino acid sequence contained in SEQ ID NO: 20 is responsible for the B box function. This 20 amino acid B box fragment has the following amino acid sequence: fkdpnapkrl psafflfcse (SEQ ID NO: 16). Another example and biologically active fragment of the B box of HMG consists of amino acids 1-20 (napkrppsaf flfcseyrpk; SEQ ID NO: 23) of SEQ ID NO: 5.

  The present invention also relates to purified preparations of antibodies that specifically bind to the B box of a vertebrate high mobility group protein (HMG) but do not specifically bind to the non-B box epitope of HMG1. In these embodiments, the antibody may inhibit the biologically active activity of the B box polypeptide, eg, the release of proinflammatory cytokines from vertebrate cells induced by HMG.

  Generating an antibody specific for an HMG B box or fragment thereof, or a cell expressing a fragment having a B box or epitope, is used as an immunogen to produce an immunospecific antibody for the immunogen. obtain. As used herein, “antibodies” include monoclonal and polyclonal antibodies, chimeric, single chain, monkeyed and humanized antibodies, and Fab fragments, including the products of Fab immunoglobulin expression libraries. .

  Since all vertebrate HMG B boxes show a high degree of sequence conservation, it is believed that any vertebrate HMG B box can induce the release of pro-inflammatory cytokines from vertebrate cells. Thus, antibodies to the B box of any vertebrate HMG are within the scope of the present invention. Preferably, the B box of HMG is a B box of mammalian HMG, more preferably a B box of mammalian HMG1, and the B box of human HMG1 provided herein as SEQ ID NO: 5 or SEQ ID NO: 20. Boxes are particularly preferred. The antibody can also be directed to a B box fragment of HMG having B box biological activity.

  Antibodies raised against B box immunogens are administered to animals, preferably non-humans, using normal protocols, B boxes, B box fragments, or cells containing B boxes or B box fragments. Can be obtained. Polypeptides, such as antigenically or immunologically equivalent derivatives or fusion proteins thereof, are used as antigens to immunize mice or other animals such as rats or chickens. A B box or fragment immunogen can be provided as a fusion protein to provide stability and increase immunogenicity of the B box or fragment. The immunogen can be coupled to an immunogen carrier protein, such as bovine serum albumin (BSA) or mussel hemocyanin (KLH), for example, by conjugation. Alternatively, a multiimmunogenic peptide comprising multiple copies of a B box or fragment may have sufficient antigenicity to improve immunogenicity, such that the use of a carrier is unnecessary. Bispecific antibodies having two antigen binding domains, each directed to a different B box epitope, can also be made by conventional methods.

  Techniques known in the art that provide antibodies produced by continuous cell line cultures can be used to prepare monoclonal antibodies. See, for example, Kohler and Milstein, Nature 256: 495-497, 1975; Kozbor et al., Immunology Today 4:72, 1983; and Cole et al., MONOCLONAL ANTIBODIES AND CANCER THERAPY, pages 77-96, Alan R. Liss, Inc., 1985. checking ...

  Techniques for producing single chain antibodies (US Pat. No. 4,946,778) can be applied to make single chain antibodies to B boxes or fragments. Also, other organisms such as transgenic mice, or other mammals can be used to express humanized antibodies.

  Where the antibody is used therapeutically for in vivo applications, the antibody is preferably modified to make it less immunogenic in the individual. For example, if the individual is a human, the antibody is preferably “humanized”; in this case, the complementarity determining region of the antibody is transferred into the human antibody (eg, Jones et al., Nature 321: 522-525, 1986; and Tempest et al., Biotechnology 9: 266-273, 1991, etc.).

  Phage display technology is also utilized to bind to polypeptides from either PCR-amplified v-gene repertoires of human lymphocytes screened to have anti-B box antibodies or from naive libraries. An antibody gene can be selected (McCafferty et al., Nature 348: 552-554, 1990; and Marks et al., Biotechnology 10: 779-783, 1992). The affinity of these antibodies can also be improved by chain shuffling (Clackson et al., Nature 352: 624-628, 1991).

  If antibodies are obtained that specifically bind to the B box epitope of HMG, they can then be screened for the ability to inhibit the release of proinflammatory cytokines without undue experimentation.

  Anti-HMG B-box antibodies that can inhibit the production of any one pro-inflammatory cytokine are within the scope of the invention. Preferably, the antibody is capable of inhibiting the production of TNF, IL-1β, or IL-6. Most preferably, the antibody is capable of inhibiting the production of any pro-inflammatory cytokine produced by vertebrate cells.

  Antibodies against HMG B box or biologically active fragments thereof are used to inhibit the release of proinflammatory cytokines from cells or to treat conditions characterized by activation of the inflammatory cytokine cascade With respect to the method, the cell can be any cell that can be induced to produce a pro-inflammatory cytokine. In preferred embodiments, the cells are immune cells (eg, macrophages, monocytes, or neutrophils). In the most preferred embodiment, the cell is a macrophage.

  In another aspect, the invention relates to a composition comprising the antibody preparation described above in a pharmaceutically acceptable excipient. In these embodiments, the composition can inhibit conditions characterized by activation of the inflammatory cytokine cascade. The conditions that can be treated with these compositions have already been listed.

  The antibody composition may also include an early sepsis mediator antagonist, as described above.

  The B box polypeptides and biologically active fragments thereof described in these embodiments are used to induce inflammatory cytokines in suitable isolated cells, in vitro or ex vivo, or as an in vivo treatment. Can be done. In any of these treatments, the polypeptide or fragment is a DNA vector or RNA encoding a B box or B box fragment having an appropriate control sequence operably linked to the encoded B box or B box fragment. It is administered by providing a vector so that a B box or B box fragment is synthesized in the treated cell or patient. In vivo applications include the use of a B box polypeptide or B box fragment polypeptide or vector as a weight loss treatment. See WO 00/47104 (the entire teachings of which are incorporated herein by reference) demonstrating that treatment with HMG1 induces weight loss. Since the B box of HMG has the activity of the HMG protein, the B box is also expected to induce weight loss. HMG B-box fragment with B-box function is also expected to induce weight loss.

  In a further aspect, the present invention also relates to a method of inhibiting the release of proinflammatory cytokines from mammalian cells. This method comprises treating cells with any HMG A-box composition or any HMG B-box or a biologically active fragment antibody composition of the HMG B-box discussed above. Including.

  This method is believed to be useful for inhibiting cytokine release from any mammalian cell that produces pro-inflammatory cytokines. However, in a preferred embodiment, the cell is a macrophage. This is because macrophage production of pro-inflammatory cytokines is associated with several serious diseases.

  This method is believed to be useful for the inhibition of any pro-inflammatory cytokine produced by mammalian cells. In a preferred embodiment, the proinflammatory cytokine is TNF, IL-1α, IL-1β, MIF or IL-6. This is because these pro-inflammatory cytokines are mediators of particularly important diseases.

  The methods of these embodiments are useful for in vitro applications (eg, in studies to determine the biological properties of pro-inflammatory cytokine production in cells). However, a preferred embodiment is in vivo therapeutic application, where the cells are in a patient suffering from or at risk for a condition characterized by activation of the inflammatory cytokine cascade.

  These in vivo aspects are believed to be useful for any condition mediated by the inflammatory cytokine cascade, including any of those listed above. Preferred conditions include appendicitis, peptic ulcer, gastric or duodenal ulcer, peritonitis, pancreatitis, ulcerative colitis, pseudomembranous colitis, acute colitis or ischemic colitis, hepatitis, Crohn's disease, asthma, allergy, anaphylactic shock , Organ ischemia, reperfusion injury, organ necrosis, hay fever, sepsis, sepsis, endotoxin shock, cachexia, septic abortion, disseminated bacteremia, burns, Alzheimer's disease, cerebral infarction, cerebral embolism, spinal cord injury , Paralysis, allograft rejection or graft versus host disease. In the most preferred embodiment, the condition is endotoxin shock or allograft rejection. Where the condition is allograft rejection, the composition can also conveniently include an immunosuppressive agent, such as cyclosporine, used to suppress allograft rejection.

  These methods may also conveniently involve administration of an early sepsis mediator antagonist. The nature of these antagonists has been discussed above.

  In yet another aspect, the invention relates to a method of treating a condition in a patient characterized by activation of an inflammatory cytokine cascade. This method can include any HMG A-box composition (including non-naturally occurring A-box polypeptides and A-box biologically active fragments), or any HMG B as discussed above. Administering to the patient a box or B box biologically active fragment antibody composition comprising a non-naturally occurring B box polypeptide or a biologically active fragment thereof. This method is expected to be useful for any condition mediated by the inflammatory cytokine cascade, including any of the conditions listed above. As with the in vivo methods described above, preferred conditions include appendicitis, peptic ulcer, gastric or duodenal ulcer, peritonitis, pancreatitis, ulcerative colitis, pseudomembranous colitis, acute colitis or ischemic colitis, Hepatitis, Crohn's disease, asthma, allergy, anaphylactic shock, organ ischemia, reperfusion injury, organ necrosis, hay fever, sepsis, sepsis, endotoxin shock, cachexia, septic abortion, disseminated bacteremia, burn, Alzheimer Disease, cerebral infarction, cerebral embolism, spinal cord injury, paralysis, allograft rejection or graft versus host disease. In the most preferred embodiment, the condition is endotoxin shock or allograft rejection. Where the condition is allograft rejection, the composition can also conveniently include an immunosuppressive agent, such as cyclosporine, used to suppress allograft rejection.

  These methods may also conveniently involve administration of an early sepsis mediator antagonist. The nature of these antagonists has been discussed above.

  In another aspect, the present invention relates to a method of stimulating the release of proinflammatory cytokines from cells. This method involves any B-box polypeptide or a biologically active B-box fragment polypeptide (eg, SEQ ID NO: 5, SEQ ID NO: 20, SEQ ID NO: 16 as described herein). Or the sequence of SEQ ID NO: 23) (including non-naturally occurring B box polypeptides and fragments). This method is useful for in vitro applications (eg, to study the effects of pro-inflammatory cytokine production on the biology of producer cells). This method is also useful for in vivo applications as discussed above (eg, performing weight loss or obesity treatment in a patient).

  Accordingly, in a further aspect, the present invention relates to a method of performing weight loss or obesity treatment in a patient. The method comprises any B-box polypeptide or B-box fragment polypeptide described herein (non-naturally occurring B-box polypeptides and fragments) in a pharmaceutically acceptable excipient. ) In an effective amount to the patient.

Screening for modulators of pro-inflammatory cytokine release from cells The present invention also relates to methods for determining whether a compound (test compound) inhibits inflammation and / or inflammatory response. The method comprises (a) a cell that releases a pro-inflammatory cytokine when exposed to a B box of vertebrate HMG or a biologically active fragment thereof, and (b) a B box or biology of HMG. Mixing the pharmaceutically active fragment thereof with the compound and then determining whether the compound inhibits the release of pro-inflammatory cytokines from the cell compared to a suitable control. In this assay, compounds that inhibit the release of pro-inflammatory cytokines are compounds that can be used to treat inflammation and / or inflammatory responses. HMG B box or biologically active HMG B box fragments can be endogenous to the cell or introduced into the cell using standard recombinant molecular biology techniques.

  Any cell that releases pro-inflammatory cytokines in response to exposure to a vertebrate HMG B box or biologically active fragment thereof in the absence of a test compound would be useful for the present invention. Is done. It is envisioned that the selected cells are important in the pathogenesis of the condition being treated with the inhibitory compound tested. For many conditions, the preferred cells are expected to be human macrophages.

  Any method of determining whether a compound inhibits the release of pro-inflammatory cytokines from cells is useful for these embodiments. A preferred method is envisioned to be a direct measurement of pro-inflammatory cytokines, for example using any number of commercially available ELISA assays. However, in some embodiments, it is preferred to measure the inflammatory effects of released cytokines, particularly when there are some pro-inflammatory cytokines produced by the test cells. As discussed above, for a number of important diseases, the predominant pro-inflammatory cytokine is TNF, IL-1α, IL-1β, MIF or IL-6; in particular TNF.

  The invention also features a method for determining whether a compound increases an inflammatory response and / or inflammation. The method comprises (a) a cell that releases a pro-inflammatory cytokine when exposed to a vertebrate HMG A box or biologically active fragment thereof; and (b) an HMG A box or biology. Mixing an active fragment thereof with a compound (test compound) and then determining whether the compound increases the release of pro-inflammatory cytokines from the cell compared to a suitable control. In this assay, a compound that decreases the release of pro-inflammatory cytokines is a compound that can be used to increase the inflammatory response and / or inflammation. The HMG A box or biologically active fragment of the HMG A box can be endogenous to the cell or can be introduced into the cell using standard recombinant molecular biology techniques. .

  Similar to the cell types useful for identifying inhibitors of inflammation described above, the release of pro-inflammatory cytokines in the vertebrate HMG A box or biologically active fragment thereof in the absence of any test compound Any cell that is normally inhibited in response to being exposed is expected to be useful for the present invention. It is envisioned that the selected cells are important in the pathogenesis of the condition being treated with the inhibitory compound tested. For many conditions, the preferred cells are expected to be human macrophages.

  Any method of determining whether a compound increases the release of pro-inflammatory cytokines from cells is useful for these embodiments. A preferred method is envisioned to be a direct measurement of pro-inflammatory cytokines, for example using any number of commercially available ELISA assays. However, in some embodiments, it is preferred to measure the inflammatory effects of released cytokines, particularly when there are some pro-inflammatory cytokines produced by the test cells. As discussed above, for a number of important diseases, the predominant pro-inflammatory cytokine is TNF, IL-1α, IL-1β, MIF or IL-6; in particular TNF.

  Preferred embodiments of the invention are described in the following examples. Other embodiments within the scope of the invention will be apparent to those skilled in the art from the detailed description or practice of the invention as disclosed herein. It is intended that the specification, along with examples and claims, be considered exemplary only.

Example 1: Materials and Methods
Cloning of HMG1 and creation of HMG1 mutants Human HMG1 clones and mutants were prepared using the following method. Recombinant full-length human HMG1 (651 base pairs; Genbank accession number U51677), the following primers: forward primer: 5'GATGGGCAAAGGAGATCCTAAG3 '(SEQ ID NO: 6) and reverse primer: 5'GCGGCCGCTTATTCATCATCATCATCTTC3' (SEQ ID NO: 7) Was cloned by PCR amplification from a human brain Quick-Clone cDNA preparation (Clontech, Palo Alto, Calif.). A variant of human HMG1 was cloned and purified as follows. A truncated form of human HMG1 was cloned by PCR amplification from a human brain Quick-Clone cDNA preparation (Clontech, Palo Alto, Calif.). The primers used were the following (forward and reverse respectively):

Carboxy terminal mutant (557 bp): 5 'GATGGGCAAAGGAGATCCTAAG 3' (SEQ ID NO: 8) and 5 'GCGGCCGCTCACTTGCTTTTTTCAGCCTTGAC3' (SEQ ID NO: 9).

Amino terminus + B box mutant (486 bp): 5'GAGCATAAGAAGAAGCACCCA3 '(SEQ ID NO: 10) and 5'GCGGCCGCTCACTTGCTTTTTTCAGCCTTGAC3' (SEQ ID NO: 11).

B box mutant (233 bp): 5'AAGTTCAAGGATCCCAATGCAAAG3 '(SEQ ID NO: 12) and 5'GCGGCCGCTCAATATGCAGCTATATCCTTTTC3' (SEQ ID NO: 13).

Amino terminus + A box mutant (261 bp): 5'GATGGGCAAAGGAGATCCTAAG3 '(SEQ ID NO: 13) and 5'TCACTTTTTTGTCTCCCCTTTGGG3' (SEQ ID NO: 14).

  A stop codon was added to each mutant to ensure protein size accuracy. The PCR product was subcloned into the pCRII-TOPO vector EcoRI site using the TA cloning method according to the manufacturer's instructions (Invitrogen, Carlsbad, CA). After amplification, the PCR product was digested with EcoRI and subcloned into the expression vector pGEX (Pharmacia) with a GST tag; the correct orientation and positive clones were confirmed by DNA sequencing on both strands. This recombinant plasmid was transformed into protease-deficient E. coli strain BL21 or BL21 (DE3) plysS (Novagen, Madison, Wis.) And fusion protein expression was achieved by isopropyl-D-thiogalactopyranoside (IPTG). Induced. Recombinant protein was obtained using affinity purification using a glutathione sepharose resin column (Pharmacia).

  The variant of HMG produced as described above has the following amino acid sequence:

Wild type HMG1:
MGKGDPKKPTGKMSSYAFFVQTCREEHKKKHPDASVNFSEFSKKCSERWKTMSAKEKGKFEDMAKADKARYEREMKTYIPPKGETKKKFKDPNAPKRLPSAFFLFCSEYRPKIKGEHPGLSIGDVAKKLGEMWNNTAADDKQPYEKKAAKLKDEKYEKDIAAEEED

Carboxy terminal mutant:
MGKGDPKKPTGKMSSYAFFVQTCREEHKKKHPDASVNFSEFSKKCSERWKTMSAKEKGKFEDMAKADKARYEREMKTYIPPKGETKKKFKDPNAPKRLPSAFFLFCSEYRPKIKGEHPGLSIGDVAKKLGEMWNNTAADDKQPYEKKAAKLKEKYEKDIAAYRAKGKPDAAKKGVEK19

B box variant:
FKDPNAPKRLPSAFFLFCSEYRPKIKGEHPGLSIGDVAKKLGEMWNNTAADDKQPYEKKAAKLKEKYEKDIAAY (SEQ ID NO: 20)

Amino terminus + A box variant:
MGKGDPKKPTGKMSSYAFFVQTCREEHKKKHPDASVNFSEFSKKCSERWKTMSAKEKGKFEDMAKADKARYEREMKTYIPPKGET (SEQ ID NO: 21), where the A box is the sequence PTGKMSSYAFFVQTCREEHKKKHPDASVNFSEFSKKCSERWKTMSAKEKGKFEDMAKADKARYEREMKTYIPK

  A polypeptide made from a GST vector lacking the HMG1 protein was included as a control (containing only the GST tag). In order to inactivate bacterial DNA that binds to wild-type HMG1 and some of its variants (carboxy terminus and B box), DNase I (Life Technologies) ), Or for wild type HMG1, benzonase nuclease (Novagen, Madison, Wis.) Was added at approximately 20 units per mL of bacterial lysate. DNA degradation was verified by ethidium bromide staining of agarose gels containing HMG1 protein before and after treatment. The protein eluate is passed through a polymyxin B column (Pierce, Rockford, IL) to remove any contaminating LPS and thoroughly dialyzed against phosphate buffered saline to obtain excess reduced glutathione. Was removed. The preparation was then lyophilized and redissolved in sterile water before use. LPS levels were less than 60 pg / μg protein for all mutants and 300 pg / μg protein for wild-type HMG-1 as measured by the horseshoe mallow-like cell lysate assay (Bio Whittaker Inc., WalkersvILle, MD) Was less than. The integrity of the protein was verified by SDS-PAGE. Recombinant rat HMG1 (Wang et al., Science 285: 248-251, 1999) was used in some experiments. This is because there are no degradation fragments as observed in purified human HMG1.

Peptide synthesis Peptides were synthesized and HPLC purified to 90% purity at the Utah State University Biotechnology Center (Logan, Utah). No endotoxin was detected in this synthetic peptide preparation as measured by the horseshoe crab assay.

Cell culture Mouse macrophage-like RAW 264.7 cells (American Type Culture Collection, RockvILle, MD) in RPMI 1640 medium supplemented with 10% fetal calf serum (Gemini, Catabasas, CA), penicillin and streptomycin (Life Technologies) (Technology, Grand Island, NY) and used at 90% confluence in Opti-MEM I medium (Life Technologies, Grand Island, NY) without serum. Polymyxin B (Sigma, St. Louis, MO) was added periodically at 100-1,000 units / mL to neutralize the activity of any contaminating LPS as described above; polymyxin B alone was trypan blue ( It did not affect cell viability as assessed by Wang et al., Supra). Polymyxin B was not used in synthetic peptide research experiments.

Measurement of TNF release from cells
TNF release was measured by standard mouse fibroblast L929 (ATCC, American Type Culture Collection, RockvILle, MD) cytotoxicity bioassay (Bianchi et al., Supra) with a minimum detectable concentration of 30 pg / mL. Recombinant mouse TNF was obtained from R & D System Inc. (Minneapolis, Minn.). Mouse fibroblast L929 cells (ATCC) in 10% fetal bovine serum (Gemini, Catabasas, CA), penicillin (50 units / mL) and streptomycin (50 μg / mL) in a humidified incubator with 5% CO ₂ ) (Life Technologies) supplemented with DMEM (Life Technologies, Grand Island, NY).

Antibody production
Polyclonal antibodies against the B box of HMG1 were raised in rabbits (Cocalico Biologicals, Inc., Reamstown, PA) and titers were assayed by immunoblotting. IgG was purified from anti-HMG1 antiserum using protein A agarose according to the manufacturer's instructions (Pierce, Rockford, IL). Anti-HMG1 B-box antibody was affinity purified by using cyanogen bromide activated Sepharose beads (Cocalico Biological, Inc.). Non-immune rabbit IgG was purchased from Sigma (St. Louis, MO). In the immunoassay, the antibody detected full length HMG1 and B box but did not cross-react with TNF, IL-1 and IL-6.

Labeling HMG1 with Na- ^125I and cell surface binding properties Using iodobeads (Pierce, Rochford, IL) according to the manufacturer's instructions, 0.2 mCi carrier-free ¹²⁵ I (NEN Life Science products Inc. , Boston, MA) and purified HMG1 protein (10 μg) was radiolabeled. Gel chromatography columns (P6 Micro Bio-Spin Chromatography Columns, Bio-Rad Laboratories, pre-equilibrated with 300 mM sodium chloride, 17.5 mM sodium citrate, pH 7.0 and 0.1% bovine serum albumin (BSA) Hercules, CA) separated ¹²⁵ I-HMG1 protein from unreacted ¹²⁵ I. The specific activity of HMG1 eluted was approximately 2.8 × 10 ⁶ cpm / μg protein. Cell surface binding studies were performed as previously described (Yang et al., Am. J. Physiol. 275: C675-C683, 1998). RAW 264.7 cells were placed on a 24-well dish and allowed to grow until confluence was reached. Cells were washed twice with ice cold PBS containing 0.1% BSA and 120 mM sodium chloride, 1.2 mM magnesium sulfate, 15 mM sodium acetate, 5 mM potassium chloride, 10 mM Tris-HCl, pH 7.4 Binding was carried out with 0.5 mL binding buffer containing 0.2% BSA, 5 mM glucose and 25,000 cpm ¹²⁵ I-HMG1 at 4 ° C. for 2 hours. At the end of the incubation, the supernatant is removed and the cells are washed three times with 0.5 mL ice-cold PBS containing 0.1% BSA, with 0.5 mL 0.5 N NaOH and 0.1% SDS at room temperature. For 20 minutes. Next, the radioactivity in the lysate was measured using a gamma ray measurement apparatus. Specific binding was determined as (total binding)-(radioactivity obtained in the presence of excess amounts of unlabeled HMG1 or A box protein).

Animal experimentation
TNF knockout mice were obtained from Amgen (Thousand Oaks, Calif.) And had a B6 × 129 history. Age-matched wild-type B6 × 129 mice were used as controls in this study. Mice were housed in a facility at the University of Florida specific pathogen-free transgenic mouse facility (Gainesville, FL) and used at 6-8 weeks of age.

  Male 6-8 week old Balb / c and C3H / HeJ mice were purchased from Harlen Sprague-Dawley (Indianapolis, IN) and acclimated for 7 days prior to use in the experiment. All animals were housed in the North Shore University Hospital Animal Facility under standard temperature and light / dark cycles.

Cecal ligation and puncture As previously described (Fink and Hard, J. Surg. Res. 49: 186-196, 1990; Wichmann et al., Crit. Care Med. 26: 2078-2086,1998; and Remick et al., Shock 4 : 89-95,1995) and cecal ligation and puncture (CLP) were performed. Briefly, Balb / c mice were anesthetized with 75 mg / kg ketamine (Fort Dodge, Fort Dodge, Iowa) and 20 mg / kg xylazine (Bohringer Ingelheim, St. Joseph, Mo.) injected into the muscle. A midline incision was made and the cecum was cut off. A 6-0 prolene suture ligature was placed at a level of 5.0 mm from the cecal tip away from the ileocecal valve.

  The ligated cecum stump was then punctured once with a 22-gauge needle so that feces were not drained directly. The cecum was then returned to the normal intraperitoneal position. The abdomen was then closed with 6-0 prolene straight sutures in two separate layers (peritoneum and fascia) to prevent fluid leakage. Normal saline solution was administered subcutaneously at 20 mL / kg body weight to reanimate all animals. Thirty minutes after the surgical procedure, each mouse received a subcutaneous injection of imipenem (0.5 mg / mouse) (Primaxin, Merck & Co., Inc., West Point, PA). The animal was then allowed to recover. Mortality was recorded up to 1 week after treatment; surviving mice were followed for 2 weeks to ensure no late deaths.

D-galactosamine sensitized mouse
D-galactosamine sensitization models have been previously described (Galanos et al., Proc. Natl. Acad. Sci. USA 76: 5939-5943, 1979; and Lehmann et al., J. Exp. Med. 165: 657-663. , 1997). Mice were injected intraperitoneally with either 20 mg D-galactosamine-HCL (Sigma) / mouse (in 200 μL PBS) and 0.1 or 1 mg HMG1 B box or vector protein (in 200 μL PBS). Daily mortality was recorded up to 72 hours post-injection; surviving mice were followed for 2 weeks with no late death due to B-box toxicity.

Spleen bacterial culture
At 24 and 30 hours after CLP, 14 mice were given either an anti-HMG1 antibody (n = 7) or a control (n = 7) as described herein and were comforted for necropsy I was killed. Spleen bacteria were harvested as previously described (Villa et al., J. Endotoxin Res. 4: 197-204, 1997). The spleen was removed using aseptic technique and homogenized in 2 mL of PBS. After serial dilution in PBS, plate the homogenate in 0.15 ml aliquots on trypsinized soy agar plates (Difco, Detroit, MI) and count the CFU after overnight incubation at 37 ° C. It was.

Statistical analysis Data are expressed as mean ± SEM unless otherwise stated. Differences between groups were determined by Student's two-tailed t test, least significant difference test after one-way ANOVA, or Fisher's exact two-tailed test.

Example 2: Mapping of HMG1 domain for promotion of cytokine activity
HMG1 has two folded DNA binding domains (A box and B box) and a negatively charged acidic carboxyl terminus. To elucidate the structural basis of HMG1 cytokine activity and to map inflammatory protein domains, we expressed the full-length and truncated forms of HMG1 by mutagenesis and monocyte cultures Purified proteins that stimulated activity in were screened (FIG. 1). A full length HMG1, a mutant lacking the carboxy terminus, a mutant containing only the B box, and a mutant containing only the A box were generated. These variants of human HMG1 are generated by polymerase chain reaction (PCR) using specific primers as described herein, and glutathione S-transferase ( The mutant protein was expressed using the GST) gene fusion system (Pharmacia Biotech, Piscataway, NJ). Briefly, DNA fragments generated by PCR were fused to a GST fusion vector and amplified in E. coli. The expressed HMG1 protein and HMG1 mutant were then isolated using a GST affinity column.

  The effect of mutants on TNF release from mouse macrophage-like RAW264.7 cells (ATCC) was demonstrated as follows. RAW 264.7 cells were cultured in RPMI 1640 medium (Life Technologies, Grand Island NY) supplemented with 10% fetal calf serum (Gemini, Catabasas, CA), penicillin and streptomycin (Life Technologies). 100 units / mL polymyxin (Sigma, St. Louis, MO) was added to suppress the activity of any contaminating LPS. Cells were incubated with 1 μg / mL full-length (wild-type) HMG1 and each HMG1 mutant protein in Opti-MEM I medium for 8 hours and conditioned supernatant (containing TNF released from the cells) The TNF released from the cells was measured by a standard mouse fibroblast L929 (ATCC) cytotoxicity bioassay (Bianchi et al., Supra) with a minimum detectable concentration of 30 pg / mL. Recombinant mouse TNF was obtained from R & D System Inc. (Minneapolis, Minn.) And used as a control in these experiments. The results of this study are shown in FIG. The data in FIG. 1 are all expressed as mean value + SEM (N = 6 to 10) unless otherwise specified.

  As shown in FIG. 1, wild-type HMG1 and carboxy-terminal truncated HMG1 significantly stimulated TNF release by monocyte cultures (mouse macrophage-like RAW264.7 cells). The B box was a strong activator of monocyte TNF release. This stimulating effect of the B box was specific. This is because the A box only slightly activated TNF release.

Example 3: HMG1 B-box protein promotes cytokine activity in a dose-dependent manner To further investigate the effect of HMG1 B-box on cytokine production, the amount of HMG1 B-box was varied to The effect of macrophage-like RAW264.7 cells on TNF, IL-1B, and IL-6 production was evaluated. As shown in FIGS. 2A-2C, RAW264.7 cells were stimulated with 8-10 μg / mL B box protein for 8 hours. Conditioned media was collected and measured for levels of TNF, IL-1β and IL-6. TNF levels are measured as described herein, and using the mouse IL-1β and IL-6 enzyme-linked immunosorbent assay (ELISA) kit (R & D System Inc., Minneapolis, Minn.) IL-1β and IL-6 levels were measured. The number of experiments was 6 or more for all experiments. The results of this study are shown in FIGS.

  As shown in FIG. 2A, the amount of TNF released from RAW264.7 cells increased as the amount of B box administered to the cells increased. As shown in FIG. 2B, the addition of 1 μg / mL or 10 μg / mL B box increased the amount of IL-β released from RAW264.7 cells. In addition, as shown in FIG. 2C, the amount of IL-6 released from RAW264.7 cells increased as the amount of B box administered to the cells increased.

  The kinetics of B box-induced TNF release were also examined. In RAW264.7 cells, TNF release and TNF mRNA expression induced between 0 and 48 hours only by B box polypeptide or GST tag polypeptide (10 μg / mL) used as control (vector) Was measured. Supernatants were analyzed for TNF protein levels by the L929 cytotoxicity assay (N = 3-5) as described herein. To measure mRNA, cells are expanded into 100 mm dishes and in Opti-MEM I medium containing only B box polypeptide or vector, as shown in FIG. 2D, 0, 4, 8, or 24 Time processed. At 4 hours, vector-only samples were evaluated. Dish cells were scraped off and total RNA was isolated by RNAzol B method according to the instructions of the manufacturing process (Tel-Test “B” Inc., Friendswood, TX). TNF (287 bp) was measured by RNase protection assay (Ambion, Austin, TX). RNA samples were stained with ethidium bromide on an agarose-formaldehyde gel to confirm that RNA was evenly added and the integrity of the RNA. The results of the RNase protection assay are shown in FIG. 2D. As shown in FIG. 2D, high levels of gene transcription resulted in activation of mononuclear leukocytes by the B box because TNF mRNA was significantly increased in mononuclear leukocytes exposed to B box protein. (FIG. 2B). TNF mRNA expression was maximal at 4 hours and decreased at 8 and 24 hours. Vector only control (GST tag) did not affect TNF mRNA expression. Release of TNF protein from RAW264.7 cells after 0, 4, 8, 24, 32, or 48 hours after administration of B box or vector (GST tag) alone, using the L929 cytotoxicity assay described herein. Similar studies were performed using and measuring. When compared to control (medium only), treatment with B box stimulated TNF protein expression (FIG. 2F), but not with vector alone (FIG. 2E). Data are representative of three independent experiments. Overall, these data suggest that the B box domain of HMG1 has cytokine activity and that this domain is responsible for the cytokine-stimulating activity of full-length HMG1.

  In summary, HMG1 B box stimulated the release of TNF, IL-1β and IL-6 from monocyte cultures in a dose-dependent manner (FIGS. 2A-2C). This is consistent with the inflammatory activity of full-length HMG1 (Andersson et al., J. Exp. Med. 192: 565-570, 2000). Furthermore, these studies suggest that the most TNF protein is released within 8 hours (FIG. 2F). This delayed pattern of TNF release is similar to the TNF release induced by HMG1 itself and is significantly slower than the TNF kinetics induced by LPS (Andersson et al., Supra).

Example 4: The first 20 amino acids of the B box of HMG1 stimulate TNF activity
The TNF-stimulating activity of HMG1 B box was further mapped. This study was conducted as follows. B box fragments were generated using synthetic peptide protection techniques as described herein. As shown in FIG. 3, five HMG1 B-box fragments containing SEQ ID NOs: HMG1 B-box amino acids 1-20, 16-25, 30-49, 45-64, or 60-74. 20 origin). RAW264.7 cells were treated with B box (1 μg / mL) or a synthetic peptide fragment of B box (10 μg / mL) as shown in FIG. 3 for 10 hours, and TNF release in the supernatant was Measurements were made as described herein. Data shown are mean ± standard error (n = 3 experiments, each experiment was performed in duplicate and confirmed using 3 separate lots of synthetic peptides). As shown in FIG. 3, TNF-stimulating activity was retained by the synthetic peptide (fkdpnapkrlpsafflfcse; SEQ ID NO: 16) corresponding to amino acids 1 to 20 of the B box of HMG1 of SEQ ID NO: 20. The strength of TNF-stimulating activity of 1-20 amino acids in length was weaker than that of either the full length of the synthetic B box (length of 1-74 amino acids) or HMG1. However, the stimulatory effect was specific because this synthetic 20 amino acid length for amino acid fragments containing 16-25, 30-49, 45-64, or 60-74 of the B box of HMG1 is This is because it did not induce release. These results are direct evidence that the B box macrophage stimulating activity is specifically located at the first 20 amino acids of the HMG B box domain of SEQ ID NO: 20. For example, the same polypeptide encoding the full length of the B box polypeptide to stimulate the release of proinflammatory cytokines or to treat a patient condition characterized by activation of the inflammatory cytokine cascade Thus, it is possible to use this B box fragment.

Example 5: HMG1 A-box protein antagonizes HMG1 inducing cytokine activity in a dose-dependent manner A weak agonist is by definition an antagonist. As shown in FIG. 1, the ability of the HMG1 A box to function as an antagonist of HMG1 activity was evaluated because the HMG1 A box only slightly induces TNF production. This study was conducted as follows. RAW264.7 cells subconfluent in 24-well dishes were cultured in Opti-MEM I medium in the presence of polymyxin B (100 units / mL) and HMG1 (1 μg / mL) and 0, 5, 10, Alternatively, it was treated with a 25 μg / mL A box for 16 hours. TNF-stimulated activity (assessed using the L929 cytotoxicity assay described herein) in samples not accepting A box is expressed as 100% and inhibition by A box is expressed as a percentage of HMG1 alone. expressed. The results of the effect of A box on TNF release from RAW264.7 cells are shown in FIG. 4A. As shown in FIG. 4A, the A box inhibited HMG1 inducing TNF release in a dose-dependent manner with an apparent EC ₅₀ of approximately 7.5 μg / mL. The data in FIG. 4A is expressed as mean ± standard deviation (n = 2-3 independent experiments).

Example 6: HMG1 A box protein inhibits cytokine activity of full length HMG1 and B box cytokine activity of HMG1 Culture of RAW 264.7 macrophages stimulated by the addition of both A box and full length HMG1 The antagonistic action of full-length HMG1 activity by the HMG1 A box or GST tag (vector control) was also measured by measuring the TNF released from. RAW 264.7 macrophage cells (ATCC) were seeded in 24-well tissue culture plates and used at 90% confluence. This cell by HMG1 and / or A box as indicated in Optimum I medium (Life Technologies, Grand Island, NY) in the presence of polymyxin B (100 units / mL, Sigma, St. Louis, MO) Was processed for 16 hours and the supernatant was collected for measuring TNF (with a mouse ELISA kit from R & D System Inc., Minneapolis, Minn.). TNF-induced activity was expressed as a percentage of the activity obtained using only HMG-1. The results of these studies are shown in FIG. 4B. FIG. 4B is a histogram of the effects of HMG1 alone, A box alone, vector (control) alone, the combination of HMG1 and A box, and the combination of HMG1 and vector. As shown in FIG. 4B, the A box of HMG1 significantly attenuated the TNF-stimulating activity of full-length HMG1.

Example 7: HMG1 A Box Protein Inhibits HMG1 Cytokine Activity by Binding to HMG1
To examine whether the HMG1 A box functions as an antagonist by displacing HMG1 binding, ¹²⁵ I-labeled-HMG1 was added to the macrophage cultures and bound at 4 ° C. 2 hours later. Was measured. Binding assays in RAW264.7 cells were performed as described herein. ¹²⁵ I-HMG1 binding was measured in RAW264.7 cells spread in 24-well dishes over the time shown in FIG. 5A. The specific binding capacity shown is equal to (all cell-associated ¹²⁵ I-HMG1 (CPM / well))-(cell-associated CPM / well in the presence of 5,000-fold molar excess of unlabeled HMG1). FIG. 5A is a graph of ¹²⁵ I-HMG1 binding versus time. As shown in FIG. 5A, HMG1 was shown to fit the first order rate equation. Specificity of binding was assessed as described in Example 1.

In addition, ¹²⁵ I-HMG-1 binding was measured in RAW264.7 cells spread on 24-well dishes and incubated with ¹²⁵ I HMG1 alone or in the presence of unlabeled HMG1 or A box. . The results of this binding assay are shown in FIG. 5B. Data are expressed as mean ± standard error from 3 separate experiments. FIG. 5B shows ¹²⁵ I-HMGB1 cell surface binding in the absence of unlabeled HMGB1 or HMGB1 (HMG1) A box, or in the presence of an unlabeled HMGB1 or HMGB1 A box in excess of 5,000 molar. Histogram, measured as a percentage of total CPM / well. In FIG. 5B, the “total” amount is equal to the count of cells associated ¹²⁵ I-HMGB1 per second (CPM) / well in the absence of unlabeled HMGB1 or A box for 2 hours at 4 ° C. . “HMGB1” or “A box” is equivalent to CPM / well of cell associated ¹²⁵ I-HMGB1 when unlabeled HMGB1 or A box is present in excess of 5,000 molar. This data is expressed as a percentage of the total count (2,382,179 CPM / well) obtained in the absence of unlabeled HMGB1 protein. These results suggest that the A box of HMG1 is a competitive antagonist of HMG1 activity in vitro that inhibits the TNF-stimulated activity of HMG1.

Example 8: Inhibition of full-length HMG1 and HMG1 B box cytokine activity by anti-B box polyclonal antibodies.
The ability of antibodies directed against the HMG1 B box to modulate the effects of full-length HMG1 or HMG1 B box was also evaluated. Affinity purified antibodies (B box antibodies) directed against the B box of HMG1 were generated as described herein and using standard techniques. To assess the effect of antibodies on HMG1-induced or HMG1 B box-induced TNF release from RAW264.7 cells, RAW264.7 cells subconfluent in 24-well dishes were treated with Anti-B box antibody (25 μg / mL or 100 μg / mL, affinity purified with antigen using HMG-1 (1 μg / mL) or HMG1 B box (10 μg / mL), Cocalico Biologicals, Inc., Reamstown, PA), or non-immune IgG added (25 μg / mL or 100 μg / mL; Sigma) and treated for 10 hours. The L929 cytotoxicity assay was used to measure TNF release from RAW264.7 cells as described herein. The results of this study are shown in FIG. This is the administration of 1 μg / mL HMG1 without administration, 1 μg / mL HMG1 + 25 μg / mL anti-B box antibody, 1 μg / mL HMG1 + 25 μg / mL IgG (control), 10 μg / mL RAW264.7 when administered with 10 mL / mL B-box, 10 μg / mL B-box + 100 μg / mL anti-B box antibody or 10 μg / mL B-box + 100 μg / mL IgG (control) 2 is a histogram of TNF released by cells. The amount of TNF released from cells induced by HMG1 alone (without adding B box antibody) was set at 100%. The data shown in FIG. 6 is the result of three independent experiments. As shown in FIG. 6, affinity purified antibodies directed against the HMG1 B box significantly inhibited TNF release induced by either full length HMG1 or HMG1 B box. These results suggest the possibility of regulating the function of HMG1 using such antibodies.

Example 9: B box protein of HMG1 is toxic to D-galactosamine-sensitized Balb / c mice
To investigate in detail whether the B box of HMG1 has cytokine activity in vivo, we study unanesthetized D-galactosamine (D-gal) sensitized Balb / c mice (cytokine toxicity) HMG1 B box protein was administered to a widely used model (Galanos et al., Supra). Briefly, D-gal (20 mg) (Sigma) and B box (0.1 mg / mL / mouse or 1 mg / mL / mouse) or GST tag (vector; 0.1 mg / mL / mouse or 1 mg / mL / mouse) Were injected intraperitoneally in mice (20-25 grams, male, Harlan Sprague-Dawley, Indianapolis, IN) as shown in Table 1. Surviving mice were monitored for up to 7 days to ensure that late death did not occur. The results of this study are shown in Table 1.

  The results of this study showed that the B box of HMG1 is lethal in a dose-dependent manner for D-galactosamine-sensitized mice. In all cases where death occurred, death occurred within 12 hours. No mortality was seen in mice treated with a comparative preparation that was a purified GST vector protein with the B box removed.

Example 10: Histology of D-galactosamine-sensitized Balb / c mice or D-galactosamine-sensitized C3H / HeJ mice dosed with HMG1 B box protein Further in vivo lethality of HMG1 B box protein To evaluate, the B box of HMG1 was again administered to D-galactosamine-sensitized Balb / c mice. D-gal (20 mg / mouse) + B box or vector (1 mg / mouse) was administered into the peritoneum of mice (3 mice per group) over 7 hours, then the neck was cut off and the mice were sacrificed. Blood was collected and organs (liver, heart, kidney, and lung) were collected and fixed with 10% formaldehyde. For histological evaluation, tissue sections were prepared by staining with hematoxylin and eosin (Criterion Inc., Vancouver, Canada). The results of these studies are shown in FIGS. These were stained with hematoxylin and eosin, kidney slices (FIG. 7A), myocardial slices (FIG. 7C), lung slices (FIG. 7E), and liver obtained from untreated mice. Scan images of the liver slices (FIGS. 7G and 7I), and kidney slices (FIG. 7B), myocardial slices (FIG. 7D), lung slices obtained from mice treated with the B box of HMG1 7B is a scan image of a specimen (FIG. 7F) and a liver section specimen (FIGS. 7H and 7J). Compared to control mice, B box treatment did not cause abnormalities in the kidneys (FIGS. 7A and 7B) and lungs (FIGS. 7E and 7F). These mice had ischemic changes and disappeared striation in the heart's myocardial fibers (FIGS. 7C and 7D as indicated by the arrows in FIG. 7D). As shown by active hepatitis (FIGS. 7G-7J), liver damage was shown to be mostly due to the B box. In FIG. 7J, hepatocyte shedding is seen surrounded by accumulated polymorphonuclear leukocytes. Arrows in FIG. 7J indicate polymorphonuclear accumulation sites (dotted lines) or apoptotic hepatocytes (solid lines). Stimulation was also provided by in vivo administration of HMG1 B box, IL-6 (315 + 93 vs 20 + 7 pg / mL, B box vs control, p <0.05) and IL-1β (15 + 3 vs. Serum levels of 4 + 1 pg / mL, B box vs. control, p <0.05) increased significantly.

  Administration of B box protein to C3H / HeJ mice (not responsive to endotoxin) was also lethal. This suggests that the HMG1 B box is lethal in the absence of LPS signaling. Lung and kidney section specimens collected 8 hours after administration of the B box and stained with hematoxylin and eosin revealed no abnormal morphological changes. However, examination of the slices from the heart revealed that there is evidence of ischemia in the myocardial fibers without the striation associated with the amorphous and light red cytoplasm. Sections from the liver showed some hepatocyte shedding and apoptosis, and a mild acute inflammatory response with occasional polymorphonuclear leukocytes. These specific pathological changes are comparable to those seen after administration of full-length HMG1, and the B box alone can reproduce a lethal pathological response to HMG1 in vivo. It is confirmed that it is possible.

  To address whether the TNF-stimulated activity of HMG1 contributes to lethal mediation by the B box, we sensitized with D-galactosamine (20 mg / mouse) and B box (1 mg / mouse, intraperitoneally) TNF knockout mice (TNF-KO, Nowak et al., Am. J. Physiol. Regul. Integr. Comp. Physiol. 278: R1202-R1209, 2000) and wild type control (B6 x 129 strains) were measured. The B box was more lethal to wild type mice (6 of 9 exposed died). However, TNF-KO mice treated with B box were not lethal (0 of 9 exposed animals died, p <0.05 vs wild type). Together with data from the RAW 264.7 macrophage cultures described herein, these data currently suggest that the B box of HMG1 confers specific cytokine activity that stimulates TNF.

Example 11: Protein levels of HMG1 are high in septic mice To investigate the role of HMG1 in sepsis, we established sepsis in mice and used the quantitative immunoassay previously described (Wang et al., Supra). Serum HMG1 was measured. Mice were subjected to a well-characterized model of sepsis, which was caused by cecal ligation and puncture (CLP), which was punctured to surgically create a cecal diverticulum. This leads to multi-bacterial infectious peritonitis and sepsis (Fink and Heard, supra; Wichmann et al., Supra; and Remick et al., Supra). The serum level of HMG1 was then measured (Wang et al., Supra). FIG. 8 is a graph showing the results of this study, in which HMG1 in mice after 0, 8, 18, 24, 48, and 72 hours subjected to CLP. The levels are explained. As shown in FIG. 8, the level of serum HMG1 did not increase significantly in the first 8 hours after puncturing the cecum. Then, it increased remarkably after 18 hours (FIG. 8). Increased serum HMG1 maintains high plateau levels for at least 72 hours after CLP and is almost identical to the previously described delay in HMG1 kinetics in endotoxemia (Wang et al., Supra). Characteristics. This temporal pattern of HMG1 release closely matched the progression of sepsis symptoms in mice. During the first 8 hours after puncturing the cecum, the animal was observed to be mildly ill, somewhat weak in activity, and unable to explore. Over the next 18 hours, the animal will be in a serious condition, will be raised and snuggled up within the group, will not seek water or food, and will not be exposed to external stimuli or examination by personnel. Only minimal reaction was shown.

Example 12: Treatment of septic mice with HMG1 A box protein increases mouse survival To determine whether HMG1 A box can inhibit HMG1 lethality during sepsis The cecum was punctured and treated by administering A box for the first 24 hours after sepsis occurred. CLP was performed on male Balb / c mice as described herein. The animals were randomly divided into groups with 15-25 mice per group. Only HMG1 A box (60 or 600 μg / mouse at each time point) or vector (GST tag, 600 μg / mouse) administered intraperitoneally twice a day for 3 days from the first 24 hours after CLP did. Survival was monitored twice a day for up to 2 weeks to ensure that late death did not occur. The results of this study are illustrated in FIG. This is a graph of the effect of 60 μg / mouse or 600 μg / mouse vector (GST; control) on survival over time (P <0.03 vs. control tested by Fisher's exact test). As shown in FIG. 9, it is clear that administration of HMG1 A box rescued mice from the lethal effects of sepsis, and survival was from vector protein (GST) with A box removed. Improved from 28% in animals treated with purified protein to 68% in animals that received the A box (P <0.03, according to Fisher's exact test). The life-saving effect of HMG1 with A-box in this sepsis model was dependent on the dose of A-box; animals treated with 600 μg / mouse of A-box became significantly more agile and active, and It was observed that feeding behavior was restored when compared to either control treated with the vector-derived preparation or animals treated with 60 μg A box alone. The latter animals remained in a severe condition, diminished activity and feeding for several days, and most died.

Example 13: Treatment of septic mice with anti-HMG1 antibody increases survival of mice Severe septic mice were also evaluated for passive immunization with anti-HMG1 antibody. In this study, male Balb / c mice (20-25 grams) were subjected to CLP as described herein. The first 24 hours after this surgical procedure, and twice a day for 3 days, 600 μg affinity purified anti-HMG1 B-box polyclonal antibody per mouse or rabbit (as a control) Of IgG was administered. Survival was monitored over 2 weeks. The results of this study are shown in FIG. 10A. This is a graph of the survival rate of septic mice treated with either control antibody or anti-HMG1 antibody. This result shows that the anti-HMG1 antibody administered to the mice 24 hours after initiating cecal puncture significantly saved the animals from death compared to the non-immune antibody administered (Fischer's exact test). P <0.02). Animals treated within 12 hours after administration of anti-HMG1 antibody showed higher activity and responsiveness compared to controls that received non-immune antibodies. Animals treated with non-immune antibodies were still snuggled, hair was distorted, and movements were slow, whereas treated animals markedly improved and normal feeding within 48 hours Resume action. In this model, anti-HMG1 antibody did not inhibit bacterial growth. Because we observed a comparable number of bacteria (CFU, aerobic colony forming unit) from the spleen 31 hours after CLP of treated animals, animals that received irrelevant antibodies (control bacteria = 3.5 This is because ± 0.9 × 10 ⁴ CFU / g; n = 7). Thereafter, the animals were monitored for up to 2 weeks and no late deaths were observed. This suggested that treatment with anti-HMG1 was completely saved from fatal sepsis and did not cause mere delayed death.

  To our knowledge, there are no other therapies targeting specific cytokines that are equally effective when given so late after the onset of sepsis. By comparison, administration of anti-TNF in this model has actually increased mortality, and anti-MIF antibodies are not useful when administered more than 8 hours after cecal puncture (Remick Supra; and Calandra et al., Nature Med. 6: 164-170, 2000). From these data, it is clear that it is possible to target HMG1 even after a long period of time, about 24 hours after cecal puncture, in order to save life from septicemia that has been established to be a fatal case Is done.

  In another example of using an anti-B box antibody to save endotoxemic mice, the ability of anti-HMG1 B box antibody to save LPS-induced septic mice was evaluated. Male Balb / c mice (20-25 grams, 26 per group) were treated with 75% lethal dose of LPS (15 mg / kg) injected intraperitoneally (IP). Anti-HMG1 B-box or non-immune rabbit serum (0.3 mL per mouse, IP at each time point) was given at 0, +12 and +24 hours after LPS administration. Mice survival was assessed over time. The results of this study are shown in FIG. 10B. This figure is a graph of the survival rate of septic mice administered anti-HMG1 B box antibody or non-immune serum. As shown in FIG. 10B, anti-HMG1 B box antibody improved the survival of septic mice.

Example 14: Inhibition of the HMG1 signaling pathway using anti-RAGE antibodies Previous data show that HMG1 receptors can mediate neurite outgrowth during brain development and smooth muscle cell migration during wound healing. RAGE has been shown to be involved (Hori et al., J. Biol. Chem. 270: 25752-25761, 1995; Merenmies et al., J. Biol. Chem. 266: 16722-16729, 1991; and Degryse Et al., J. Cell Biol. 152: 1197-1206, 2001). We have HMG1 (1 μg / mL), LPS (0.1 μg / mL), or HMG1 B box (1 μg / mL) in the presence of anti-RAGE antibodies (25 μg / mL) or non-immune IgG (25 μg / mL). ) Was used to measure TNF release in RAW 264.7 cultures stimulated. Briefly, the cells were seeded in 24-well tissue culture plates and used at 90% confluence. LPS (E. coli 0111: B4, Sigma, St Louis, MO) was sonicated for 20 minutes before use. As shown in FIG. 11A, HMG1 (1 μg / mL) in serum-free Opti-MEM I medium (Life Technologies) in the presence of anti-RAGE antibody (25 μg / mL) or non-immune IgG (25 μg / mL). ), LPS (0.1 μg / mL), or HMG1 B-box (1 μg / mL) for 16 hours and measure TNF using the L929 cytotoxicity assay described herein In order to do so, the supernatant was collected. Prior to use, IgG-purified polyclonal anti-RAGE antibodies (Cat. No. sc-8230, N-16, Santa Cruz Biotech, Inc., Santa Cruz, Calif.) Were dialyzed extensively against PBS. The results of this study are shown in FIG. 11A. This figure is a histogram of the effect of HMG1, LPS, or HMG1 B box in the presence of anti-RAGE antibodies or non-immune IgG (control) on TNF release from RAW264.7 cells. As shown in FIG. 11A, anti-RAGE antibody significantly inhibited B box-induced TNF release of HMG1 compared to non-immune IgG. This suppression was specific because anti-RAGE did not significantly inhibit LPS-stimulated TNF release. Notably, the greatest inhibitory effect of anti-RAGE is to reduce HMG-1 signaling by only 40%, suggesting that other signaling pathways may be involved in HMG1 signaling .

  To examine the effect of HMG1 or HMG1 B box on the NF-kB-dependent ELAM promoter, the following experiment was performed. Luciferase reporter under the control of an NF-kB-dependent ELAM promoter in addition to an expression plasmid or empty vector encoding the mouse MyD88-dominant negative (DN) variant (corresponding to amino acids 146-296) The plasmid was used to perform transient cotransfection of RAW 264.7 macrophages as described by Means et al. (J. Immunol. 166: 4074-4082, 2001). A portion of the cells was then stimulated for 5 hours with full length HMG1 (100 ng / mL) or purified HMG1 B box (10 μg / mL). Cells were then harvested and luciferase activity was measured using standard methods. All transfections were performed in triplicate and repeated at least three times, and only one representative experiment is shown in FIG. 11B. As shown in FIG. 11B, HMG1 stimulates luciferase activity in samples not co-transfected with MyD88 dominant negative, and in samples co-transfected with MyD88 dominant negative, The level of stimulation decreased. This result was also seen in the samples to which the HMG B box was administered.

The effect of HMG1 or B box of HMG1 on the activation of NF-kB was also examined. CHO reporter cell lines that constitutively express human toll-like receptor 2 (TLR2) or human toll-like receptor 4 (TLR4) have already been described (Means et al., J. Immunology, 163: 3920-3927, 1999). These reporter systems also contain a stably transfected ELAM-CD25 reporter gene and express human CD25 on their surface as a result of NF-kB activation. CHO / TLR2 and CHO / TLR4 cells were stimulated over 18 hours by IL-1 (10 ng / mL), purified full length HMG-1 (100 ng / mL), or purified B box (10 μg / mL) . Following stimulation, cells were stained with PE-labeled anti-CD25 monoclonal antibody and CD25 surface expression was measured by flow cytometry. The results of this study are shown in FIG. 11C. Percentage of CD25 ⁺ cells in the unstimulated and stimulated cell population that was retained to exclude the lower 5% of cells based on average FL1 fluorescence (activity The data is expressed as (magnification rate). In CHO / TLR4 cells, stimulation with each of the HMG1 and HMG1 B boxes reduced CD25 expression compared to the CHO / TLR2 sample.

  The effect of anti-RAGE antibody, anti-TLR2 antibody, combination of anti-RAGE antibody and anti-TLR2 antibody or IgG on HMG-1-mediated TNF release in RAW264.7 cells was also examined. RAW264.7 cells were seeded in 24-well tissue culture plates and used at 90% confluence. An anti-RAGE antibody (catalog number sc-8230, Santa Cruz Biotech, Inc., Santa Cruz, CA), anti-TLR2 antibody (affinity purified polyclonal antibody, catalog number sc-12504, D17, Santa Cruz Biotech, Inc.) or Optimum I medium (Life Technologies) containing polymyxin B (100 units / mL, Sigma, St. Louis, MO) with or without IgG (non-immune IgG, Sigma, St. Louis, MO) , Grand Island, NY), cell incubation with HMG-1 was performed for 16 hours. Prior to use, the antibody was dialyzed against PBS to remove sodium azide. Conditioned media was collected and TNF ELISA performed using standard ELISA methods. Data (n = 3) were expressed as the percent of activity obtained with HMG-1 alone. The results of this study are shown in FIG. 11D. Both anti-RAGE and anti-TLR2 antibodies significantly (* P <0.05) inhibited HMG-1-mediated TNF release. IgG was irrelevant, while the combination of the two antibodies showed an additive effect on the inhibition of TNF release.

  The invention has been shown and described in detail with reference to preferred embodiments. It will be appreciated by those skilled in the art that various shapes and details may be changed without departing from the spirit of the invention as encompassed by the appended claims.

FIG. 1 is a schematic representation of HMG1 variants and their activity (pg / mL) for TNF release. FIG. 2A shows the effect of 0 μg / mL, 0.01 μg / mL, 0.1 μg / mL, 1 μg / mL, or 10 μg / mL B box on TNF release (pg / mL) in RAW264.7 cells. It is a histogram. FIG. 2B shows the effect of 0 μg / mL, 0.01 μg / mL, 0.1 μg / mL, 1 μg / mL, or 10 μg / mL B box on IL-1β release (pg / mL) in RAW264.7 cells. It is a histogram which shows. FIG. 2C shows the effect of 0 μg / mL, 0.01 μg / mL, 0.1 μg / mL, 1 μg / mL, or 10 μg / mL B box on IL-6 release (pg / mL) in RAW264.7 cells. It is a histogram which shows. FIG. 2D is a scan image of an RNase protection assay blot, B box (at time 0, 4, 8 or 24 hours after administration) or vector (at time 4 hours after administration). Only shows the effect of TNF on mRNA expression in RAW264.7 cells. FIG. 2E shows TNF protein release (pg / mL) from RAW264.7 cells at 0, 4, 8, 24, 32, or 48 hours after administration of HMG1 B box. It is a histogram of the effect given to. FIG. 2F shows the effect on TNF protein release (pg / mL) from RAW264.7 cells at 0 hours, 4 hours, 8 hours, 24 hours, 32 hours, or 48 hours after administration of the vector. This is a histogram. FIG. 3 is a schematic representation of HMG1 B box mutants and their activity (pg / mL) for TNF release. FIG. 4A shows the effect of 0 μg / mL, 5 μg / mL, 10 μg / mL, or 25 μg / mL HMG1 A-box protein on TNF released from RAW264.7 cells (only HMG1-mediated TNF release). Graph (as a percentage). FIG. 4B shows that HMG1 (0 or 1.5 μg / mL), HMG1 A box (0 or 10 μg / mL), or vector (0 or 10 μg / mL) alone, or a combination thereof, was released from RAW264.7 cells. Is a histogram of the effect on TNF (as a percentage of HMG1-mediated TNF release alone). FIG. 5A is a graph of ¹²⁵ I-HMGB1 binding (CPM / well) binding to RAW264.7 cells over a long period (minutes). FIG. 5B shows 2 hours at 4 ° C. in the absence of unlabeled HMGB1 or HMG1 A box (total amount), or unlabeled HMGB1 (HMGB1) or A box (A box) in excess of 5,000 molar. Histogram of ¹²⁵ I-HMGB1 binding as measured as a percentage of the total amount of CPM per well. FIG. 6 shows HMG-1 (0 μg / mL or 1 μg / mL) or HMG1 B box (0 μg / mL or 10 μg / mL) alone, or anti-B box antibody (25 μg / mL or 100 μg / mL) or Histogram of the effect on TNF release from RAW264.7 cells, combined with IgG (25 μg / mL or 100 μg / mL) (expressed as a percentage of HMG1-mediated TNF release only). FIG. 7A is a scan image of a kidney section obtained from an untreated mouse stained with hematoxylin and eosin. FIG. 7B is a scan image of a kidney section obtained from a mouse administered with a B box of HMG1 stained with hematoxylin and eosin. FIG. 7C is a scan image of a myocardium section sample obtained from an untreated mouse stained with hematoxylin and eosin. FIG. 7D is a scan image of a myocardium section sample obtained from a mouse administered with the B box of HMG1 stained with hematoxylin and eosin. FIG. 7E is a scanned image of lung sections obtained from untreated mice stained with hematoxylin and eosin. FIG. 7F is a scan image of a lung section obtained from a mouse administered HMG1 B box stained with hematoxylin and eosin. FIG. 7G is a scan image of a liver section sample obtained from an untreated mouse stained with hematoxylin and eosin. FIG. 7H is a scan image of a liver section sample obtained from a mouse administered with a B box of HMG1 stained with hematoxylin and eosin. FIG. 7I is a scan image (high magnification) of a liver slice obtained from an untreated mouse stained with hematoxylin and eosin. FIG. 7J is a scan image (high magnification) of a liver section sample obtained from a mouse administered with a B box of HMG1 stained with hematoxylin and eosin. FIG. 8 is a graph of HMGB1 levels (ng / mL) with respect to time (hours) in mice subjected to cecal ligation and puncture (CLP). FIG. 9 is a graph with respect to time (days) of the effect of A box (60 μg / mouse or 600 μg / mouse) or untreated on survival of mice after cecal ligation and puncture (CLP). FIG. 10A is a graph regarding the time (days) of the effect of anti-HMG1 antibody (black circle) or untreated (white circle) on the survival of mice after cecal ligation and puncture (CLP). FIG. 10B is a graph of the effect of anti-HMG1 B-box antiserum (black squares) or untreated ( ^* ) on the survival (days) of mice administered lipopolysaccharide (LPS). FIG. 11A shows TNF release from RAW264.7 cells in which anti-RAGE antibodies or non-immune IgG were treated with HMG1 (HMG-1), lipopolysaccharide (LPS), or HMG1 B box (B box). It is a histogram of the influence which it has on. FIG. 11B shows that stimulation of HMG1 or HMG1 B-box polypeptide is a mouse MyD88-dominant negative (+ MyD 88 DN) mutant (which corresponds to amino acids 146-296) or an empty vector (-MyD 88 DN ) Is a histogram of the effect on activation (measured by luciferase activity) of the NFkB-dependent ELAM promoter in RAW264.7 cells co-transfected with. Data are expressed as the percentage of the average value of luciferase (minus background) (fold of activation) + standard deviation from unstimulated and stimulated cells. FIG. 11C shows that a CHO reporter cell line constitutively expressing human TLR2 (white bar) or human TLR4 (black bar) stimulated by IL-1, HMG1, or HMG1 B box, It is a histogram of the influence which it has on the expression of CD25. Percentage of percent of CD25 ⁺ cells in the unstimulated and stimulated cell population that was retained to exclude the lower 5% of cells based on average FL1 fluorescence (activity The data is expressed as (magnification rate). FIG. 11D shows that administration of anti-RAGE antibody, anti-TLR2 antibody, anti-RAGE antibody and anti-TLR2 antibody together or IgG is measured in RAW264.7 cells (measured as percent TNF release without antibody). HMG1—Histogram of effects on mediated TNF release. FIG. 12A is the amino acid sequence of human HMG1 polypeptide (SEQ ID NO: 1). FIG. 12B is the amino acid sequence of rat and mouse HMG1 (SEQ ID NO: 2). FIG. 12C is the amino acid sequence of human HMG2 (SEQ ID NO: 3). FIG. 12D is the amino acid sequence (SEQ ID NO: 4) of the A box polypeptide of human, mouse, and rat HMG1. FIG. 12E is the amino acid sequence of human, mouse, and rat HMG1 B box polypeptides (SEQ ID NO: 5). FIG. 12F is the nucleic acid sequence (SEQ ID NO: 6) of the forward primer for human HMG1. FIG. 12G is the nucleic acid sequence (SEQ ID NO: 7) of the reverse primer for human HMG1. FIG. 12H is the nucleic acid sequence of the forward primer (SEQ ID NO: 8) for the carboxy terminal variant of human HMG1. FIG. 12I is the nucleic acid sequence of the reverse primer (SEQ ID NO: 9) for the carboxy terminal variant of human HMG1. FIG. 12J is the nucleic acid sequence of the forward primer for the amino terminus + B box variant of human HMG1 (SEQ ID NO: 10). FIG. 12K is the nucleic acid sequence of the reverse primer for the amino terminus + B box variant of human HMG1 (SEQ ID NO: 11). FIG. 12L is the nucleic acid sequence of the forward primer (SEQ ID NO: 12) for the B box variant of human HMG1. FIG. 12M is the reverse primer nucleic acid sequence (SEQ ID NO: 13) for the B box variant of human HMG1. FIG. 12N is the nucleic acid sequence of the forward primer for the amino terminus + A box variant of human HMG1 (SEQ ID NO: 14). FIG. 12O is the nucleic acid sequence of the reverse primer for the amino terminus + A box variant of human HMG1 (SEQ ID NO: 15). FIG. 13 is an alignment of HMG1 polypeptide sequences from rat (SEQ ID NO: 2), mouse (SEQ ID NO: 2), and human (SEQ ID NO: 18).

Claims (24)

  Vertebrate high mobility group protein (HMG) A boxes or non-naturally occurring HMG A boxes that can inhibit the release of proinflammatory cytokines from vertebrate cells treated with high mobility group (HMG) proteins A polypeptide comprising   Vertebrate high mobility group protein (HMG) A box biologically active fragment or naturally occurring that can inhibit the release of proinflammatory cytokines from vertebrate cells treated with high mobility group (HMG) protein A polypeptide that is a non-existent HMG A box biologically active fragment.   Vertebrate high mobility group protein (HMG) capable of inhibiting the release of pro-inflammatory cytokines from vertebrate cells treated with high mobility group (HMG) protein in a pharmaceutically acceptable excipient ) A composition comprising a polypeptide comprising an A box or a non-naturally occurring HMG A box.   Vertebrate high mobility group protein (HMG) capable of inhibiting the release of pro-inflammatory cytokines from vertebrate cells treated with high mobility group (HMG) protein in a pharmaceutically acceptable excipient ) A composition comprising a polypeptide that is an A-box biologically active fragment or a non-naturally occurring HMG A-box biologically active fragment.   Specific binding to vertebrate high mobility group protein (HMG) B box but specific to non-B box epitopes of HMG that can inhibit the release of proinflammatory cytokines from vertebrate cells treated with HMG Purified preparation of antibodies that do not bind manually.   Specific to vertebrate high mobility group protein (HMG) B box in a pharmaceutically acceptable excipient that can inhibit the release of pro-inflammatory cytokines from vertebrate cells treated with HMG A composition comprising a purified preparation of an antibody that binds but does not specifically bind to a non-B box epitope of HMG.   A polypeptide comprising a vertebrate high mobility group protein (HMG) B box or a non-naturally occurring HMG B box, but not full length HMG, capable of inducing release of proinflammatory cytokines from vertebrate cells.   A polypeptide that is a vertebrate high mobility group protein (HMG) B box biologically active fragment or a non-naturally occurring HMG B box biologically active fragment.   A polypeptide comprising a vertebrate high mobility group protein (HMG) B box or a non-naturally occurring HMG B box, but not full length HMG, capable of inducing release of proinflammatory cytokines from vertebrate cells. The vector to encode.   Vertebrate high mobility group protein (HMG) B box biologically active fragment or non-naturally occurring HMG B box biologically active fragment capable of inducing release of proinflammatory cytokines from vertebrate cells A vector encoding a polypeptide.   Treating mammalian cells with an amount of a purified preparation of an antibody that specifically binds to a vertebrate high mobility group protein (HMG) B box but not specifically to a non-B box epitope of HMG. A method of inhibiting the release of proinflammatory cytokines from mammalian cells.   Vertebrate that can inhibit the release of proinflammatory cytokines from vertebrate cells treated with an amount of high mobility group (HMG) protein sufficient to inhibit the release of proinflammatory cytokines from vertebrate cells Of inhibiting the release of pro-inflammatory cytokines from mammalian cells, comprising treating mammalian cells with a polypeptide comprising a high mobility group protein (HMG) A box or a non-naturally occurring HMG A box .   Vertebrate that can inhibit the release of proinflammatory cytokines from vertebrate cells treated with an amount of high mobility group (HMG) protein sufficient to inhibit the release of proinflammatory cytokines from vertebrate cells Treating a mammalian cell with a polypeptide polypeptide that is a high mobility group protein (HMG) A box biologically active fragment or a non-naturally occurring HMG A box biologically active fragment of Of inhibiting the release of pro-inflammatory cytokines from the body.   An amount of an antibody that specifically binds to a vertebrate high mobility group protein (HMG) B box but does not specifically bind to a non-B box epitope of HMG in an amount sufficient to inhibit the proinflammatory cytokine cascade. A method of treating a condition in a patient characterized by activation of an inflammatory cytokine cascade comprising administering a purified preparation to the patient.   Vertebrate that can inhibit the release of proinflammatory cytokines from vertebrate cells treated with an amount of high mobility group (HMG) protein sufficient to inhibit the release of proinflammatory cytokines from vertebrate cells Treating a condition in a patient characterized by activation of the inflammatory cytokine cascade, comprising administering to the patient a polypeptide comprising a high mobility group protein (HMG) A box or a non-naturally occurring HMG A box Method.   Vertebrate that can inhibit the release of proinflammatory cytokines from vertebrate cells treated with an amount of high mobility group (HMG) protein sufficient to inhibit the release of proinflammatory cytokines from vertebrate cells Of an inflammatory cytokine cascade comprising administering to a patient a polypeptide that is a high mobility group protein (HMG) A box biologically active fragment or a non-naturally occurring HMG A box biologically active fragment of A method of treating a condition in a patient having characteristics.   Contains a vertebrate high mobility group protein (HMG) B box or a non-naturally occurring HMG B box sufficient to stimulate the release of proinflammatory cytokines from the cell, but not full-length HMG A method of stimulating the release of proinflammatory cytokines from a cell comprising treating the cell with a polypeptide.   A vertebrate high mobility group protein (HMG) B box biologically active fragment or a non-naturally occurring HMG B box biological activity in an amount sufficient to stimulate the release of proinflammatory cytokines from the cell A method of stimulating the release of proinflammatory cytokines from a cell comprising treating the cell with a polypeptide that is a fragment.   Contains a vertebrate high mobility group protein (HMG) B box or a non-naturally occurring HMG B box sufficient to stimulate the release of proinflammatory cytokines from the cell, but not full-length HMG A method of stimulating the release of proinflammatory cytokines from a cell comprising treating the cell with a vector encoding a polypeptide.   A vertebrate high mobility group protein (HMG) B box biologically active fragment or a non-naturally occurring HMG B box biological activity in an amount sufficient to stimulate the release of proinflammatory cytokines from the cell A method of stimulating the release of proinflammatory cytokines from a cell comprising treating the cell with a vector encoding a polypeptide that is a fragment.   Contains a vertebrate high mobility group protein (HMG) B box or a non-naturally occurring HMG B box in an amount sufficient to stimulate the release of pro-inflammatory cytokines from the cell. A method of causing weight loss or treating obesity in a patient, comprising administering to the patient an effective amount of a polypeptide not included.   A vertebrate high mobility group protein (HMG) B box biologically active fragment or a non-naturally occurring HMG B box biological activity in an amount sufficient to stimulate the release of proinflammatory cytokines from the cell A method of causing weight loss or treating obesity in a patient comprising administering to the patient an effective amount of a polypeptide that is a fragment. Binding the compound to (a) a cell that releases a pro-inflammatory cytokine when exposed to a vertebrate high mobility group protein (HMG) B box; and (b) binding the HMG B box; Determining whether to inhibit the release of pro-inflammatory cytokines from the cells,
A method of determining whether a compound inhibits inflammation. A compound and (a) a cell that releases pro-inflammatory cytokines when exposed to a vertebrate high mobility group protein (HMG) B box or biologically active fragment thereof; and (b) an HMG B box or organism thereof. Binding a biologically active fragment and then determining whether the compound inhibits the release of proinflammatory cytokines from the cell.
A method of determining whether a compound inhibits inflammation.

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